Diagnosis of a neuroautoimmune disease

ABSTRACT

The present invention relates to a method for diagnosing a disease comprising the step detecting in a sample comprising antibodies from a patient an autoantibody binding to a polypeptide selected from the group comprising NSF, STX1B, DNM1 and VAMP2, a polypeptide comprising a polypeptide selected from the group comprising NSF, STX1B, DNM1 and VAMP2, or a variant thereof, a use of said polypeptide for the diagnosis of a disease, an autoantibody binding to a polypeptide selected from the group comprising NSF, STX1B, DNM1 and VAMP2, a use of the autoantibody for the diagnosis of a disease, a method for isolating an autoantibody binding to a polypeptide selected from the group comprising NSF, STX1B, DNM1 and VAMP2, a pharmaceutical composition or medical device comprising said polypeptide according to the present invention, a kit for the diagnosis of a disease comprising said polypeptide or said medical device and a use of said polypeptide or autoantibody for the manufacture of a kit or medical device.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 310159_412_SEQUENCE_LISTING.txt. The text fileis 71 KB, was created on Jun. 12, 2018, and is being submittedelectronically via EFS-Web.

The present invention relates to a method for diagnosing a diseasecomprising the step detecting in a sample comprising antibodies from apatient an autoantibody binding to a polypeptide selected from the groupcomprising NSF, STX1B, DNM1 and VAMP2, a polypeptide comprising apolypeptide selected from the group comprising NSF, STX1B, DNM1 andVAMP2, or a variant thereof, a use of said polypeptide for the diagnosisof a disease, an autoantibody binding to a polypeptide selected from thegroup comprising NSF, STX1B, DNM1 and VAMP2, a use of the autoantibodyfor the diagnosis of a disease, a method for isolating an autoantibodybinding to a polypeptide selected from the group comprising NSF, STX1B,DNM1 and VAMP2, a pharmaceutical composition or medical devicecomprising said polypeptide according to the present invention, a kitfor the diagnosis of a disease comprising said polypeptide or saidmedical device and a use of said polypeptide or autoantibody for themanufacture of a kit or medical device.

Developing diagnostic systems for neurological diseases is a continuingchallenge in biomedical science, not in the least because many symptomsencountered may be accounted for by a huge variety of causes includinggenetically-inherited diseases, drug abuse, malnutrition, infection,injury, psychiatric illness, immunological defects and cancer.

Since a neurological disease is rarely associated with a uniquecharacteristic pattern of clinical symptoms, it is often difficult toprovide a reliable diagnosis solely based on the observation andexamination of the patients affected or their medical history.

The importance of an early diagnosis cannot be overemphasized. Manyneurological disorders, most prominently Alzheimer's and Parkinson'sdiseases as well as Multiple Sclerosis, cannot be cured, but drugs areavailable that may be used to slow down their progression. In addition,certain rare types of cancer are associated with neurological symptoms.The earlier the diagnosis, the better the chances to exploit thespectrum of available therapies to the full benefit of the patient.

This holds all the more true in the case of neurological diseasesassociated with autoantibodies. In some cases, the link between aspecific detectable autoantibody and a condition is sufficiently strongto allow for an immediate diagnosis.

But even if it is not, the detection of autoantibodies may point thephysician in charge to therapeutic means that may be used to amelioratethe patient's condition. There is a variety of widely usedimmunosuppressants that may be used regardless of the nature of theautoantibody's target. Alternatively, apheresis may be used to removeautoantibodies from the patient's blood. In many cases, patients went onto lead a normal life following early diagnosis and treatment of aneurological autoimmune disease.

Diagnostic assays based on the detection of autoantibodies may alsocorroborate the diagnosis of diseases other than those associated withautoantibodies. If it turns out that a blood sample is devoid ofspecific autoantibodies, this is likely to help the physician in chargeexclude a range of possibilities and thus narrow down the spectrum ofplausible conditions.

Examples of neurological conditions coinciding with the emergence ofautoantibodies include Neuromyelitis optica, a disease characterized byloss of visual perception and spinal cord function, and anti-NMDAreceptor encephalitis, which is associated with autonomic dysfunction,hypoventilation, cerebellar ataxia, hemiparesis, loss of consciousness,or catatonia. Whilst the involvement of autoantibodies and the nature ofthese conditions as such was previously poorly understood, many of thisdisease can now be diagnosed and treated efficiently owing to theavailability of assays based on the detection of autoantibodies.

Therefore, it is paramount that new approaches be developed todistinguish neurological conditions associated with autoantibodies fromthose that are not.

WO1997/021729 and U.S. Pat. No. 5,693,476 disclose the use of NSF,syntaxins and VAMP proteins as part of artificially formed complexes toidentify substances that modulate synaptic transmission but do notdisclose the existence, let alone diagnostical usefulness of anautoantibody binding to a polypeptide selected from the group comprisingNSF, STX1B, DNM1 and VAMP2.

Nicot et al. report decreased transcript and protein levels of STX1B inmurine experimental autoimmune encephalitis (Nicot A, Ratnakar P V, RonY, Chen C C, Elkabes S. Regulation of gene expression in experimentalautoimmune encephalomyelitis indicates early neuronal dysfunction.Brain. 2003 February; 126(Pt 2):398-412), but do not disclose theexistence, let alone diagnostical usefulness of autoantibody binding toa polypeptide selected from the group comprising NSF, STX1B, DNM1 andVAMP2.

Hirai et al. report autoantibodies against VAMP2 in 21% of patients withtype 1 diabetes (Hirai H, Miura J, Hu Y, Larsson H, Larsson K, LernmarkA, lvarsson SA, Wu T, Kingman A, Tzioufas A G, Notkins A L. Selectivescreening of secretory vesicle-associated proteins for autoantigens intype 1 diabetes: VAMP2 and NPY are new minor autoantigens. Clin Immunol.2008 June; 127(3):366-74) by an in vitro-transcription/translationimmunoprecipitation protocol directed at secretory vesicle-associatedproteins. In the same report, the authors disclose that they did notdetect any antibodies against STX1A, a protein with 97.6% homology toSTX1B. They do not disclose the existence, let alone diagnosticalusefulness of an autoantibody binding to a polypeptide selected from thegroup comprising NSF, DNM1 and STX1B. Furthermore, the autoantibodybinding to VAMP2 was not considered a marker for diagnosing neurologicaldisorders.

The problem underlying the present invention is to provide novelreagents, devices and methods that may be used to support the diagnosisand treatment of an autoimmune disease, preferably an autoimmune diseaseof the nervous system or associated with a neurological disease orneurological symptoms, more preferably selected from the groupcomprising stiff-person syndrome and encephalitis, preferablyencephalitis.

Another problem underlying the present invention is to provide novelreagents, devices and methods that may be used to distinguish autoimmunediseases, in particular neurological autoimmune diseases, morepreferably selected from the group comprising stiff-person syndrome andencephalitis, preferably encephalitis, from diseases other thanautoimmune diseases, not in the least to determine the most promisingtreatment regimen, more specifically whether or not an immunosuppressivetreatment is adequate.

The problem underlying the present invention is solved by thesubject-matter of the attached independent and dependent claims.

In a first aspect, the problem is solved by a method for diagnosing adisease comprising the step detecting in a sample comprising antibodiesfrom a patient an autoantibody binding to a polypeptide selected fromthe group comprising NSF, STX1B, DNM1 and VAMP2.

In a second aspect, the problem is solved by a polypeptide comprising apolypeptide selected from the group comprising NSF, STX1B, DNM1 andVAMP2 or a variant thereof, which is preferably immobilized, morepreferably on a solid carrier.

In a third aspect, the problem is solved by a use of a polypeptideaccording to the present invention for the diagnosis of a disease,preferably comprising the step detecting in a sample an autoantibodybinding to a polypeptide selected from the group comprising NSF, STX1B,DNM1 and VAMP2.

In a 4^(th) aspect, the problem is solved by the polypeptide accordingto the present invention for use in a treatment of a disease.

In a 5^(th) aspect, the problem is solved by an autoantibody, preferablyan isolated autoantibody binding to a polypeptide selected from thegroup comprising NSF, STX1B, DNM1 and VAMP2, wherein the antibody ispreferably in complex with the polypeptide according to the presentinvention.

In a 6^(th) aspect, the problem is solved by a use of the autoantibodyaccording to the present invention for the diagnosis of a disease.

In a 7^(th) aspect, the problem is solved by a method for isolating anautoantibody binding to a polypeptide selected from the group comprisingNSF, STX1B, DNM1 and VAMP2, comprising the steps

-   -   a) contacting a sample comprising the autoantibody with a        polypeptide according to the present invention under conditions        compatible with formation of a complex, wherein said        autoantibody binds to said polypeptide,    -   b) isolating the complex formed in step a),    -   c) dissociating the complex isolated in step b) and    -   d) separating the autoantibody from the polypeptide.

In an 8^(th) aspect, the problem is solved by a pharmaceuticalcomposition or medical device, preferably diagnostic device, comprisingthe polypeptide according to the present invention.

In a 9^(th) aspect the problem is solved by a kit for the diagnosis of adisease, which kit comprises the polypeptide according to the presentinvention or the medical device according to the present invention,wherein preferably the kit comprises in addition a means for detecting acomplex comprising the polypeptide according to the present inventionand/or an antibody binding to a polypeptide selected from the groupcomprising NSF, STX1B, DNM1 and VAMP2.

In another aspect, the method according to the present invention is amethod for calibrating a diagnostic test system or for ascertaining thereliability and/or sufficient capacity of such a test system or atherapeutic system for removing autoantibodies from the blood of apatient. In the case of a diagnostic test system, autoantibodies are notdetected in a sample from a patient to be diagnosed, but are detected inan artificial solution of known composition, in particular comprising adefined concentration of autoantibody or a recombinant antibody ofdefined concentration which binds to the autoantigen. This artificialsolution can be used as a positive control. The term “calibrating”, asused herein, can be understood as using an antibody binding to apolypeptide selected from the group comprising NSF, STX1B, DNM1 andVAMP2 or a variant thereof on the diagnostic test system to obtainqualitative, semi-quantitative or quantitative data of the antibodybinding to a corresponding antigen. Preferably, the antigen may be apolypeptide selected from the group comprising NSF, STX1B, DNM1 andVAMP2 or a variant expressed in a cell of a tissue section or a celltransfected with a nucleic acid molecule comprising the geneticinformation to express the antigen of interest. The diagnostic systemmay be any system that allows the detection of autoantibodies in asample, such as a medical or diagnostic device according to the presentinvention.

In the case of a therapeutic system, for example an apparatus forapharesis, the method may be used to develop such a system and test itsreliability and/or efficiency and/or capacity. For example, following anapharesis run or prior to starting an apharesis run, a solutioncomprising a defined concentration of an antibody binding to thepolypeptide of the present invention may be contacted with the system,and the method according to the present invention may be used toconfirming that the system is capable of depleting the solution of theantibody.

In a preferred embodiment, the patient has or the disease is associatedwith one or more, preferably two or more symptoms selected from thegroup comprising progressive stiffness in truncal muscles, includingthoracolumbar paraspinal and abdominal muscles, abdominal wall musclesand proximal leg, rigid gait, lumbar hyperlordosis, chronic pain, spasmsin proximal limb and axial muscles, sensitivity to touch and sound,hyperekplexia, myoclonus, depression, anxiety, phobia, fever, headache,confusion, dysarthria, dysphagia, nystagmus, oscillopsia, vertigo,nausea, ataxia, paraesthesia, muscle wasting, dizziness, seizures,epilepsy and tremor.

In a 10^(th) aspect, the problem is solved by a use of a polypeptideaccording to the present invention or the autoantibody according to thepresent invention or an antibody to a polypeptide from the groupcomprising NSF, STX1B, DNM1 and VAMP2 or the medical device according tothe present invention for the manufacture of a kit, medical device,preferably diagnostic device, preferably for the diagnosis of a disease.

In a preferred embodiment, the disease is a neurological disease,preferably an autoimmune disease of the nervous system, more preferablyselected from the group comprising stiff-person syndrome andencephalitis, preferably encephalitis. In a preferred embodiment, themethod or use according to the present invention is intended todetermine whether the disease, preferably neurological disease, has anautoimmune component, preferably one amenable to immunosuppressivetreatment.

In a preferred embodiment, the sample is a bodily fluid comprisingantibodies, preferably selected from the group comprising whole blood,serum, cerebrospinal fluid and saliva.

In a preferred embodiment, the autoantibody or complex is detected usinga method selected from the group comprising immunodiffusion techniques,immunoelectrophoretic techniques, light scattering immunoassays,agglutination techniques, labeled immunoassays such as those from thegroup comprising radiolabeled immunoassay, enzyme immunoassays, morepreferably ELISA, chemiluminscence immunoassays, and immunofluorescence,preferably indirect immunofluorescence.

In a preferred embodiment, the medical device is selected from the groupcomprising a glass slide, preferably for microscopy, a biochip, amicrotiter plate, a test strip, a membrane, preferably a line blot, achromatography column and a bead, preferably a magnetic bead.

In a preferred embodiment, the autoantibody or complex is detected usinga method selected from the group comprising immunodiffusion techniques,immunoelectrophoretic techniques, light scattering immunoassays,agglutination techniques, labeled immunoassays such as those from thegroup comprising radiolabeled immunoassay, enzyme immunoassays, morepreferably ELISA, chemiluminscence immunoassays, and immunofluorescence,preferably indirect immunofluorescence.

The present invention is based on the inventors' surprising finding thatan autoantibody to NSF, an autoantibody to STX1B, an autoantibody toDNM1 and an autoantibody to VAMP2 exist and may be detected in samplesfrom a number of patients suffering from neurological symptoms, but notin samples obtained from healthy subjects.

Furthermore, the present invention is based on the inventors' surprisingfinding that the novel neurological disease may be diagnosed by the wayof detection of an autoantibody to a polypeptide selected from the groupcomprising NSF, STX1B, DNM1 and VAMP2

Without wishing to be bound to any theory, the presence of suchautoantibodies suggests that activity and function of one or more thanone polypeptide selected from the group comprising NSF, STX1B, DNM1 andVAMP2 and/or downstream effectors is impaired in patients having suchautoantibodies to the effect that neurological symptoms occur.

N-ethylmaleimide sensitive fusion protein (NSF), syntaxin 1B (STX1B),dynamin 1 (DNM1) and vesicle-associated membrane protein 2 (VAMP2) areintracellular peripheral membrane proteins highly expressed in thebrain, especially in the cell body and axons of neurons (Hong W, Lev STethering the assembly of SNARE complexes. Trends Cell Biol. 2014 24:35-43).

They are part of SNARE (Soluble NSF Attachment Protein Receptor)complexes that are involved in the docking and/or fusion of synapticvesicles with the presynaptic membrane in neurons. Thereby, NSF, STX1B,DNM1 and VAMP2, respectively, modulate neurotransmitter release,including release of gamma-amino butyric acid (GABA) and glycine frominhibitory neurons (Südhof TC, Rizo J. Synaptic vesicle exocytosis. ColdSpring Harb Perspect Biol. 2011 Dec. 1; 3(12). pii: a005637).

Cleavage of STX1B and VAMP2 by botulinum toxin from Clostridiumbotulinum, consisting of several proteases designated as botulinumneurotoxin A-G, abolishes the release of the neurotransmitteracetylcholine from axon endings at the neuromuscular junction and thuscauses flaccid paralysis. Similarly, cleavage of VAMP2 by tetanus toxinfrom Clostridium tetani leads to lockjaw characterized by muscle spams.

NSF is a 82 kDa polypeptide containing 744 amino acids. It is requiredfor vesicle-mediated transport. It catalyzes the fusion of transportvesicles within the Golgi cisternae. It is also required for transportfrom the endoplasmic reticulum to the Golgi stack. It seems to functionas a fusion protein required for the delivery of cargo proteins to allcompartments of the Golgi stack independent of vesicle origin.

STX1B is a 33 kDa polypeptide containing 288 amino acids. It ispotentially involved in docking of synaptic vesicles at presynapticactive zones.

VAMP2 is a 13 kDa polypeptide containing 116 amino acids. It is involvedin the targeting and/or fusion of transport vesicles to their targetmembrane. Modulates the gating characteristics of the delayed rectifiervoltage-dependent potassium channel KCNB1.

STX1B and VAMP2 are part of the SNARE core complex in neurons.

DNM1 is a 97 kDa polypeptide containing 864 amino acids. Said proteinpossesses mechanochemical properties used to tubulate and severmembranes, and is involved in clathrin-mediated endocytosis and othervesicular trafficking processes. Actin and other cytoskeletal proteinsact as binding partners for DNM1, which can also self-assemble leadingto stimulation of GTPase activity.

The present invention relates to a polypeptide comprising a mammalian,preferably human polypeptide selected from the group comprising NSF,STX1B, DNM1 and VAMP2, or antigenic variants reactive to autoantibodiesbinding to NSF, STX1B, DNM1 or VAMP2, respectively. Mammalian NSF,STX1B, DNM1 and VAMP2 include those from human, monkey, mouse, rat,rabbit, guinea pig or pig and are preferably human NSF, STX1B, DNM1 andVAMP2.

In a more preferred embodiment, NSF is the polypeptide encoded by thedata base codes P46459-1 or P46459-2, preferably P46459-1. The data basecodes of the corresponding cDNA are NM_006178 (NCBI), respectively.Throughout this application, any data base codes cited refers to theUniprot data base, more specifically the version on the filing date ofthis application or its earliest priority application.

In a more preferred embodiment, STX1B is the polypeptide encoded by database codes P61266-1 or P61266-2, preferably P61266-1. The data basecodes of the corresponding cDNA are NM_052874 (NCBI), respectively.

In a more preferred embodiment, DNM1 is the polypeptide encoded by database codes Q05193, preferably Q05193-1 (UniProt). The data base codes ofthe corresponding cDNA are NM_004408 (NCBI), respectively.

In a more preferred embodiment, VAMP2 is the polypeptide encoded by database code P63027-1. The data base codes of the corresponding cDNA areNM_014232 (NCBI), respectively.

The teachings of the present invention may not only be carried out usingpolypeptides, in particular a polypeptide comprising the native sequenceof a polypeptide selected from the group comprising NSF, STX1B, DNM1 andVAMP2 or nucleic acids having the exact sequences referred to in thisapplication explicitly, for example by function, name, sequence oraccession number, or implicitly, but also using variants of suchpolypeptides or nucleic acids.

In a preferred embodiment, the term “variant”, as used herein, may referto at least one fragment of the full length sequence referred to, morespecifically one or more amino acid or nucleic acid sequence which is,relative to the full-length sequence, truncated at one or both terminiby one or more amino acids. Such a fragment comprises or encodes for apeptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150 or200 successive amino acids of the original sequence or a variantthereof. The total length of the variant may be at least 6, 7, 8, 9, 10,11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids.

In another preferred embodiment, the term “variant” relates not only toat least one fragment, but also to a polypeptide or a fragment thereofcomprising amino acid sequences that are at least 40, 50, 60, 70, 75,80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to the referenceamino acid sequence referred to or the fragment thereof, wherein aminoacids other than those essential for the biological activity, forexample the ability of an antigen to bind to an (auto)antibody, or thefold or structure of the polypeptide are deleted or substituted and/orone or more such essential amino acids are replaced in a conservativemanner and/or amino acids are added such that the biological activity ofthe polypeptide is preserved. The state of the art comprises variousmethods that may be used to align two given nucleic acid or amino acidsequences and to calculate the degree of identity, see for exampleArthur Lesk (2008), Introduction to bioinformatics, Oxford UniversityPress, 2008, 3^(rd) edition. In a preferred embodiment, the ClustalWsoftware (Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R.,McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm,A., Lopez, R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007).Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) isused using default settings.

In a preferred embodiment, the variant is a linear, non-foldedpolypeptide, which is optionally denatured.

In a preferred embodiment, the polypeptide and variants thereof may, inaddition, comprise chemical modifications, for example isotopic labelsor covalent modifications such as glycosylation, phosphorylation,acetylation, decarboxylation, citrullination, methylation, hydroxylationand the like. The person skilled in the art is familiar with methods tomodify polypeptides. Any modification is designed such that it does notabolish the biological activity of the variant.

Moreover, variants may also be generated by fusion with other knownpolypeptides or variants thereof and comprise active portions ordomains, preferably having a sequence identity of at least 70, 75, 80,85, 90, 92, 94, 95, 96, 97, 98 or 99% when aligned with the activeportion of the reference sequence, wherein the term “active portion”, asused herein, refers to an amino acid sequence, which is less than thefull length amino acid sequence or, in the case of a nucleic acidsequence, codes for less than the full length amino acid sequence,respectively, and/or is a variant of the natural sequence, but retainsat least some of the biological activity.

In a preferred embodiment, the term “variant” of a nucleic acidcomprises nucleic acids the complementary strand of which hybridizes,preferably under stringent conditions, to the reference or wild typenucleic acid. Stringency of hybridization reactions is readilydeterminable by one of ordinary skilled in the art, and in general is anempirical calculation dependent on probe length, washing temperature andsalt concentration. In general longer probes require higher temperaturesfor proper annealing, while shorter probes less so. Hybridizationgenerally depends on the ability of denatured DNA to reanneal tocomplementary strands present in an environment below their meltingtemperature: The higher the degree of desired homology between the probeand hybridizable sequence, the higher the relative temperature which maybe used. As a result, higher relative temperatures would tend to makethe reaction conditions more stringent, while lower temperature less so.For additional details and explanation of stringency of hybridizationreactions, see Ausubel, F. M. (1995), Current Protocols in MolecularBiology. John Wiley & Sons, Inc. Moreover, the person skilled in the artmay follow the instructions given in the manual Boehringer Mannheim GmbH(1993) The DIG System Users Guide for Filter Hybridization, BoehringerMannheim GmbH, Mannheim, Germany and in Liebl, W., Ehrmann, M., Ludwig,W., and Schleifer, K. H. (1991) International Journal of SystematicBacteriology 41: 255-260 on how to identify DNA sequences by means ofhybridization. In a preferred embodiment, stringent conditions areapplied for any hybridization, i.e. hybridization occurs only if theprobe is 70% or more identical to the target sequence. Probes having alower degree of identity with respect to the target sequence mayhybridize, but such hybrids are unstable and will be removed in awashing step under stringent conditions, for example lowering theconcentration of salt to 2×SSC or, optionally and subsequently, to0.5×SSC, while the temperature is, in order of increasing preference,approximately 50° C.-68° C., approximately 52° C.-68° C., approximately54° C.-68° C., approximately 56° C.-68° C., approximately 58° C.-68° C.,approximately 60° C.-68° C., approximately 62° C.-68° C., approximately64° C.-68° C., approximately 66° C.-68° C. In a particularly preferredembodiment, the temperature is approximately 64° C.-68° C. orapproximately 66° C.-68° C. It is possible to adjust the concentrationof salt to 0.2×SSC or even 0.1×SSC. Nucleic acid sequences having adegree of identity with respect to the reference or wild type sequenceof at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% may beisolated. In a preferred embodiment, the term variant of a nucleic acidsequence, as used herein, refers to any nucleic acid sequence thatencodes the same amino acid sequence and variants thereof as thereference nucleic acid sequence, in line with the degeneracy of thegenetic code.

The variant of the polypeptide has biological activity. In a preferredembodiment, such biological activity is the ability to bind to anautoantibody binding to the respective polypeptide selected from thegroup comprising NSF, STX1B, DNM1 and VAMP2, as found in a patientsuffering from an autoimmune disease associated with such autoantibody,preferably associated with a neurological disease such as stiff-personsyndrome, paraneoplastic stiff-person syndrome, progressiveencephalomyelitis with rigidity and myoclonus and encephalitiy,preferably stiff-person syndrome associated with such an autoantibody.For example, whether or not a variant of NSF has such biologicalactivity may be checked by determining whether or not the variant ofinterest binds to an autoantibody from a sample of a patient whichautoantibody binds to wild type NSF, preferably as determined by Westernblotting using recombinant protein as described in the experimentalsection of this application. Whether or not a variant of STX1B has suchbiological activity may be checked by determining whether or not thevariant of interest binds to an autoantibody from a sample of a patientwhich autoantibody binds to wild type STX1B, preferably as determined byindirect immunofluorescence with mammalian cells expressing STX1B asdescribed in the experimental section of this application. Whether ornot a variant of DNM1 has such biological activity may be checked bydetermining whether or not the variant of interest binds to anautoantibody from a sample of a patient which autoantibody binds to wildtype DNM1, preferably as determined by indirect immunofluorescence withmammalian cells expressing DNM1 as described in the experimental sectionof this application. Whether or not a variant of VAMP2 has suchbiological activity may be checked by determining whether or not thevariant of interest binds to an autoantibody from a sample of a patientwhich autoantibody binds to wild type VAMP2, preferably as determined byindirect immunofluorescence with mammalian cells expressing VAMP2 asdescribed in the experimental section of this application.

The polypeptide according to the present invention, which comprises apolypeptide selected from the group comprising NSF, STX1B, DNM1 andVAMP2 or a variant thereof, including the autoantibody according to thepresent invention, when used to carry out the teachings of the presentinvention, may be provided in any form and at any degree ofpurification, from liquid samples, tissues or cells comprising saidpolypeptide in an endogenous form, more preferably cells overexpressingthe polypeptide, crude or enriched lysates of such cells, to purifiedand/or isolated polypeptide which is optionally essentially pure. In apreferred embodiment, the polypeptide is a native polypeptide, whereinthe term “native polypeptide”, as used herein, refers to a foldedpolypeptide, more preferably to a folded polypeptide purified fromtissues or cells, more preferably from mammalian cells or tissues,optionally from non-recombinant tissues or cells. In another preferredembodiment, the polypeptide is a recombinant protein, wherein the term“recombinant”, as used herein, refers to a polypeptide produced usinggenetic engineering approaches at any stage of the production process,for example by fusing a nucleic acid encoding the polypeptide to astrong promoter for overexpression in cells or tissues or by engineeringthe sequence of the polypeptide itself. The person skilled in the art isfamiliar with methods for engineering nucleic acids and polypeptidesencoded (for example, described in Sambrook, J., Fritsch, E. F. andManiatis, T. (1989), Molecular Cloning, CSH or in Brown T. A. (1986),Gene Cloning—an introduction, Chapman & Hall) and for producing andpurifying native or recombinant polypeptides (for example Handbooks“Strategies for Protein Purification”, “Antibody Purification”,“Purifying Challenging Proteins” (2009/2010), published by GE HealthcareLife Sciences, and in Burgess, R. R., Deutscher, M. P. (2009), Guide toProtein Purification). In a preferred embodiment, a polypeptide is pureif at least 60, 70, 80, 90, 95 or 99 percent of the polypeptide in therespective sample consists of said polypeptide as judged by SDSpolyacrylamide gel electrophoresis followed by Coomassie blue stainingand visual inspection.

If the inventive polypeptide is provided in the form of tissue, it ispreferred that the tissue is mammalian tissue, for example human, rat,primate, donkey, mouse, goat, horse, sheep, pig or cow, more preferablybrain tissue, most preferably cerebellum. If a cell lysate is used, itis preferred that the cell lysate comprises the membranes associatedwith the surface of the cell or is in fact a fraction enriched inmembranes. If said polypeptide is provided in the form of a recombinantcell, it is preferred that the recombinant cell is a eukaryotic cellsuch as a yeast cell, more preferably a cell from a multicellulareukaryote such as a plant, mammal, frog or insect, most preferably froma mammal, for example rat, human, primate, donkey, mouse, goat, horse,sheep, pig or cow.

The polypeptide used to carry out the inventive teachings, including anyvariants, is preferably designed such that it comprises at least oneepitope recognized by and/or binds specifically to the autoantibodybinding to a polypeptide selected from the group comprising NSF, STX1B,DNM1 and VAMP2. Any epitope is more preferably an epitope recognized bysuch an autoantibody only, by contrast to antibodies other than anautoantibody to NSF, DNM1, STX1B or VAMP2. In one embodiment, suchepitope comprises a stretch of 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40,50, 60, 70, 80, 90, 100 or more, preferably at least 9 but no more than16, consecutive amino acids from NSF, STX1B, DNM1 and VAMP2,respectively. The person skilled in the art is familiar with guidelinesused to design peptides having sufficient immunogenicity, for examplethose described in Jackson, D. C., Fitzmaurice, C. J., Brown, L. E.,Zeng, W. (1999), Preparation and properties of totally syntheticimmunogenes, Vaccine Volume 18, Issues 3-4, September 1999, Pages355-361; and Black, M., Trent, A., Tirrell, M. and Olive, C. (2010),Advances in the design and delivery of peptide subunit vaccines with afocus on Toll-like receptor agonists, Expert Rev Vaccines, 2010February; 9(2): 157-173. Briefly, it is desirable that the peptide meetsas many as possible of the following requirements: (a) it has a highdegree of hydrophilicity, (b) it comprises one or more residues selectedfrom the group comprising aspartate, proline, tyrosine andphenylalanine, (c) is has, for higher specificity, no or little homologywith other known peptides or polypeptides, (d) it needs to besufficiently soluble and (e) it comprises no glycosylation orphosphorylation sites unless required for specific reasons.Alternatively, bioinformatics approaches may be followed, for examplethose described by Moreau, V., Fleury, C., Piquer, D., Nguyen, C.,Novali, N., Villard, S., Laune, D., Granier, C. and Molina, F. (2008),PEPOP: Computational design of immunogenic peptides, BMC Bioinformatics2008, 9:71. If the polypeptide is STX1B or a variant thereof, theepitope is preferably in SEQ ID NO: 5.

The inventive polypeptide, which comprises a polypeptide selected fromthe group comprising NSF, STX1B, DNM1 and VAMP2 or a variant thereof,when used according to the present invention, may be provided in anykind of conformation. For example, the polypeptide may be an essentiallyunfolded, a partially or a fully folded polypeptide. In a preferredembodiment, the polypeptide is folded in the sense that the epitopesessential for the binding to the inventive autoantibody, or the proteinor variant thereof in its entirety, adopt the fold adopted by the nativeprotein in its natural environment. The person skilled in the art isfamiliar with methods suitable to determine whether or not a polypeptideis folded and if it is, which structure it has, for example limitedproteolysis, NMR spectroscopy, CD spectroscopy or X-ray crystallography(see for example Banaszak L. J. (2008), Foundations of StructuralBiology, Academics Press, or Teng Q. (2013), Structural Biology:Practical Applications, Springer), preferably CD spectroscopy is used.

The inventive polypeptide may be a fusion protein which comprises aminoacid sequences other than those taken from NSF, STX1B, DNM1 and VAMP2,in particular a C-terminal or N-terminal tag, preferably a C-terminaltag, which is, in a preferred embodiment, as used herein, an additionalsequence motif or polypeptide having a function that has some biologicalor physical function and may, for example, be used to purify,immobilize, precipitate or identify the inventive polypeptide. In a morepreferred embodiment, the tag is a sequence or domain capable of bindingspecifically to a ligand, for example a tag selected from the groupcomprising His tags, thioredoxin, maltose binding protein,glutathione-S-transferase, a fluorescence tag, for example from thegroup comprising green fluorescent protein.

The inventive polypeptide may be an immobilized polypeptide. In apreferred embodiment, the term “immobilized”, as used herein, refers toa molecule bound to a solid carrier insoluble in an aqueous solution,more preferably via a covalent bond, electrostatic interactions,encapsulation or entrapment, for example by denaturing a globularpolypeptide in a gel, or via hydrophobic interactions, most preferablyvia one or more covalent bonds. Various suitable carriers, for examplepaper, polystyrene, metal, silicon or glass surfaces, microfluidicchannels, membranes, beads such as magnetic beads, column chromatographymedia, biochips, polyacrylamide gels and the like have been described inthe literature, for example in Kim, D., and Herr, A. E. (2013), Proteinimmobilization techniques for microfluidic assays, Biomicrofluidics7(4), 041501. This way, the immobilized molecule, together with theinsoluble carrier, may be separated from an aqueous solution in astraightforward manner, for example by filtration, centrifugation ordecanting. An immobilized molecule may be immobilized in a reversible orirreversible manner. For example, the immobilization is reversible ifthe molecule interacts with the carrier via ionic interactions that canbe masked by addition of a high concentration of salt or if the moleculeis bound via a cleavable covalent bond such as a disulphide bridge whichmay be cleaved by addition of thiol-containing reagents. By contrast,the immobilization is irreversible if the molecule is tethered to thecarrier via a covalent bond that cannot be cleaved in aqueous solution,for example a bond formed by reaction of an epoxide group and an aminegroup as frequently used to couple lysine side chains to affinitycolumns. The protein may be indirectly immobilized, for example byimmobilizing an antibody or other entity having affinity to themolecule, followed by formation of a complex to the effect that themolecule-antibody complex is immobilized. Various ways to immobilizemolecules are described in the literature, for example in Kim, D., Herr,and A. E. (2013), Protein immobilization techniques for microfluidicassays, Biomicrofluidics 7(4), 041501. In addition, various reagents andkits for immobilization reactions are commercially available, forexample from Pierce Biotechnology.

It is essential that the sample used for the diagnosis in line with thedetection of autoantibodies according to the present invention comprisesantibodies, also referred to as immunoglobulins. Typically the sample ofa bodily fluid comprises a representative set of the entirety of thesubject's immunoglobulins. However, the sample, once provided, may besubjected to further processing which may include fractionation,centrifugation, enriching or isolating the entirety of immunoglobulinsor any immunoglobulin class of the subject, which may affect therelative distribution of immunoglobulins of the various classes.

The reagents, devices, methods and uses described throughout thisapplication may be used for the diagnosis of a disease. In a preferredembodiment, the disease is a neurological disease. In a more preferredembodiment, the term “neurological disease”, as used herein, refers toany disease associated with a defect of the nervous system, in anotherpreferred embodiment, the term “PNS”, abbreviation of paraneoplasticneurological syndrome, as used herein, refers to a systemic disorderindirectly caused by the presence of a tumor, for example, as a resultof the production release of substances such as hormones or cytokinesnot normally produced by the cell of origin of the tumor or are producedat increased concentration or the production and release of biologicallyactive cells. The tumor may be too small for detection.

In a preferred embodiment, the term “diagnosis”, as used herein, refersto any kind of procedure aiming to obtain information instrumental inthe assessment whether a patient suffers or is likely or more likelythan the average or a comparative subject, the latter preferably havingsimilar symptoms, to suffer from certain a disease or disorder in thepast, at the time of the diagnosis or in the future, to find out how thedisease is progressing or is likely to progress in the future or toevaluate the responsiveness of a patient with regard to a certaintreatment, for example the administration of immunosuppressive drugs. Inother words, the term “diagnosis” comprises not only diagnosing, butalso prognosticating and/or monitoring the course of a disease ordisorder.

In many cases the mere detection, in other words determining whether ornot detectable levels of the antibody are present in the sample, issufficient for the diagnosis. If the autoantibody can be detected, thiswill be information instrumental for the clinician's diagnosis andindicates an increased likelihood that the patient suffers from adisease. In a preferred embodiment, the autoantibody is deemeddetectable if it can be detected using one or more methods selected fromthe group comprising immunoprecipitation, indirect immunofluorescence,ELISA or line blot, preferably immunoprecipitation. Experimental detailsare as described in the experimental section of this application or asin text books or practical manuals as available at the priority date ofthis application. In a preferred embodiment, the relative concentrationof the antibody in the serum, compared to the level that may be found inthe average healthy subject, may be determined. While in many cases itmay be sufficient to determine whether or not autoantibodies are presentor detectable in the sample, the method carried out to obtaininformation instrumental for the diagnosis may involve determiningwhether the concentration is at least 0.1, preferably 0.2, 0.5, 1, 2, 5,10, 20, 25, 50, 100, 200, 500, 1000, 10000 or 100000 times higher thanthe concentration found in the average healthy subject. In a preferredembodiment, the relative concentration of the autoantibody is determinedusing one or more methods selected from the group comprisingsemi-quantitative immunoprecipitation, semi-quantitativesemi-quantitative indirect immunofluorescence, ELISA orsemi-quantitative line blot, preferably ELISA. Experimental details areas described in the experimental section of this application or as intext books or practical manuals as available at the priority date ofthis application.

The person skilled in the art will appreciate that a clinician doesusually not conclude whether or not the patient suffers or is likely tosuffer from a disease, condition or disorders solely on the basis of asingle diagnostic parameter, but needs to take into account otheraspects, for example the presence of other autoantibodies, markers,blood parameters, clinical assessment of the patient's symptoms or theresults of medical imaging or other non-invasive methods such aspolysomnography, to arrive at a conclusive diagnosis. See Baenkler H. W.(2012), General aspects of autoimmune diagnostics, in Renz, H.,Autoimmune diagnostics, 2012, de Gruyter, page 3. The value of adiagnostic agent or method may also reside the possibility to rule outone disease, thus allowing for the indirect diagnosis of another. In apreferred embodiment, the meaning of any symptoms or diseases referredto throughout this application is in line with the person skilled in theart's understanding as of the filing date or, preferably, earliestpriority date of this application as evidenced by text books andscientific publications.

Therefore, the term “diagnosis” does preferably not imply that thediagnostic methods or agents according to the present invention will bedefinitive and sufficient to finalize the diagnosis on the basis of asingle test, let alone parameter, but may refer to a contribution towhat is referred to as a “differential diagnosis”, i. e. a systematicdiagnostic procedure considering the likelihood of a range of possibleconditions on the basis of a range of diagnostic parameters.Consequently, the inventive method, polypeptide or use, optionally fordetermining whether a patient suffers from the a disease, may compriseobtaining a sample from a patient, preferably a human patient,determining whether an autoantibody binding to a polypeptide selectedfrom the group comprising NSF, STX1B, DNM1 and VAMP2 is present in saidsample, wherein said determining is performed by contacting the samplewith the inventive polypeptide and detecting whether binding occursbetween said polypeptide and said autoantibody, preferably using alabeled secondary antibody, wherein said autoantibody binds to saidpolypeptide if present in the sample, and diagnosing the patient assuffering or being more likely to suffer from said neurological disorderif the autoantibody was determined to be present in the sample.

In a preferred embodiment, the method according to the present inventioncomprises detecting more than one autoantibody from the group comprisingan autoantibody to each of the polypeptides NSF, STX1B, DNM1, VAMP2,GAD65, GAD67, IA-2, ZNT8 and amphiphysin. In a more preferredembodiment, this may involve a) detecting an autoantibody from the groupcomprising an autoantibody to each of the polypeptides GAD65, GAD67,IA-2, ZNT8 and amphiphysin, preferably GAD65 and GAD67 and b) detectingan autoantibody from the group comprising an autoantibody to each of thepolypeptides NSF, STX1B, DNM1, VAMP2, preferably NSF.

The term “diagnosis” may also refer to a method or agent used todistinguish between two or more conditions associated with similar oridentical symptoms.

The term “diagnosis” may also refer to a method or agent used to choosethe most promising treatment regime for a patient. In other words, themethod or agent may relate to selecting a treatment regimen for asubject. For example, the detection of autoantibodies may indicate thatan immunosuppressive therapy is to be selected, which may includeadministrating to the patient one or more immunosuppressive drugs.

The present invention relates to a complex comprising an antibody,preferably autoantibody, binding to the inventive polypeptide. Such acomplex may be used or detected as part of a method for diagnosing adisease. A liquid sample comprising antibodies from a subject may beused to practice the method if autoantibodies to a polypeptide selectedfrom the group comprising NSF, STX1B, DNM1 and VAMP2 are to be detected.Such a liquid sample may be any bodily fluid comprising a representativeset of antibodies from the subject, preferably a sample comprisingantibodies of the IgG immunoglobulin class from the subject. Forexample, a sample may be cerebrospinal fluid (CSF), blood or bloodserum, lymph, insterstitial fluid and is preferably serum or CSF, morepreferably serum.

The step contacting a liquid sample comprising antibodies with theinventive polypeptide(s) may be carried out by incubating an immobilizedform of said polypeptide(s) in the presence of the sample comprisingantibodies under conditions that are compatible with the formation ofthe complex comprising the respective polypeptide and an antibody,preferably an autoantibody, binding to the inventive polypeptide. Theliquid sample, then depleted of antibodies binding to the inventivepolypeptide(s) may be removed subsequently, followed by one or morewashing steps. Finally the complex comprising the antibody or antibodiesand the polypeptide(s) may be detected. In a preferred embodiment, theterm “conditions compatible with the formation of the complex” areconditions that allow for the specific antigen-antibody interactions tobuild up the complex comprising the polypeptide an the antibody. In apreferred embodiment such conditions may comprise incubating thepolypeptide in sample diluted 1:100 in PBS buffer for 30 minutes at 25°C. In a preferred embodiment, the term “autoantibody”, as used herein,refers to an antibody binding specifically to an endogenous molecule ofthe animal, preferably mammal, which produces said autoantibody, whereinthe level of such antibody is more preferably elevated compared to theaverage healthy person or person not suffering from the disease,preferably healthy person. In a most preferred embodiment, theautoantibody is an autoantibody binding to a polypeptide selected fromthe group comprising NSF, STX1B, DNM1 and VAMP2.

The method according to the present invention is preferably an in vitromethod.

In a preferred embodiment, the detection of the complex for theprognosis, diagnosis, methods or test kit according to the presentinvention comprises the use of a method selected from the groupcomprising immunodiffusion techniques, immunoelectrophoretic techniques,light scattering immunoassays, agglutination techniques, labeledimmunoassays such as those from the group comprising radiolabeledimmunoassay, enzyme immunoassays, preferably ELISA, chemiluminscenceimmunoassays, and immunofluorescence, preferably indirectimmunofluorescence techniques. The person skilled in the art is familiarwith these methods, which are also described in the state of the art,for example in Zane, H. D. (2001), Immunology—Theoretical & PracticalConcepts in Laboratory Medicine, W. B. Saunders Company, in particularin Chapter 14.

Alternatively, a sample comprising tissue comprising the inventivepolypeptide rather than a liquid sample may be used. The tissue sampleis preferably from a tissue expressing endogenous polypeptide selectedfrom the group comprising NSF, STX1B, DNM1 and VAMP2, preferably at anincreased level compared to the average tissue in the respectiveorganism's, preferably human body. Such a sample, which may be in theform of a tissue section fixed on a carrier, for example a glass slidefor microscopic analysis, may then be contacted with the inventiveantibody, preferably autoantibody, binding to the inventive polypeptide.The antibody is preferably labeled to allow for distinction fromendogenous antibodies binding to the inventive polypeptide, so thatnewly formed complexes may be detected and, optionally, quantified. Ifthe amount of complexes formed is lower than the amount found in asample taken from a healthy subject, the subject from whom the sampleexamined has been taken is likely to suffer from a disease.

Any data demonstrating the presence or absence of the complex comprisingthe antibody and the inventive polypeptide may be correlated withreference data. For example, detection of said complex indicates thatthe patient who provided the sample analyzed has suffered, is sufferingor is likely to suffer in the future from a disease. If a patient hasbeen previously diagnosed and the method for obtaining diagnosticallyrelevant information is run again, the amount of complex detected inboth runs may be correlated to find out about the progression of thedisease and/or the success of a treatment. For example, if the amount ofcomplex is found to increase, this suggests that the disorder isprogressing, likely to manifest in the future and/or that any treatmentattempted is unsuccessful.

In a preferred embodiment, a microtiterplate, membrane, blot such as dotblot or line blot is used to carry out the diagnostic method accordingto the invention. The person skilled in the art is familiar with theexperimental setup, which is described in the state of the art (Raoult,D., and Dasch, G. A. (1989), The line blot: an immunoassay formonoclonal and other antibodies. Its application to the serotyping ofgram-negative bacteria. J. Immunol. Methods, 125 (1-2), 57-65;WO2013041540).

In another preferred embodiment, the prognosis, diagnosis, methods ortest kit in line with the inventive teachings contemplate the use ofindirect immunofluorescence. The person skilled in the art is familiarwith such techniques and the preparation of suitable samples, which aredescribed in the state of the art (U.S. Pat. No. 4,647,543; Voigt, J.,Krause, C., Rohwäder, E, Saschenbrecker, S., Hahn, M., Danckwardt, M.,Feirer, C., Ens, K, Fechner, K, Barth, E, Martinetz, T., and Stöcker, W.(2012), Automated Indirect Immunofluorescence Evaluation of AntinuclearAutoantibodies on HEp-2 Cells,” Clinical and Developmental Immunology,vol. 2012, doi:10.1155/2012/65105; Bonilla, E., Francis, L., Allam, F.,et al., Immuno-fluorescence microscopy is superior to fluorescent beadsfor detection of antinuclear antibody reactivity in systemic lupuserythematosus patients, Clinical Immunology, vol. 124, no. 1, pp. 18-21,2007). Suitable reagents, devices and software packages are commerciallyavailable, for example from EUROIMMUN, Lübeck, Germany.

A sample may be subjected to a test to determine only whether anautoantibody binding to polypeptide selected from the group comprisingNSF, STX1B, DNM1 and VAMP2 is present, but it is preferred thatdiagnostic methods, tests, devices and the like contemplate determiningthe presence of autoantibodies to one or more additional polypeptides,preferably related to neurological autoimmune diseases, preferablyselected from the group comprising Hu, Yo, Ri, CV2, PNMA1, PNMA2,DNER/Tr, ARHGAP26, ITPR1, ATP1A3, NBC1, Neurochrondrin, CARPVIII, Zic4,Sox1, Ma, MAG, MP0, MBP, GAD65, amphiphysin, recoverin, GABA A receptor(EP13189172.3), GABA B receptor (EP2483417), glycine receptor, gephyrin,IgLON5 (2016/0349275), DPPX (US2015/0247847), aquaporin-4, MOG, NMDAreceptor, AMPA receptors, GRM1, GRM5, LGI1, VGCC and mGluR1 and CASPR2,which antigens are preferably immobilized, for example on a medicaldevice such as a line blot. In a more preferred embodiment, anautoantibody to a polypeptide selected from the group comprising NSF,STX1B, DNM1 and VAMP2, and an autoantibody to GAD65 is detected. Thediagnostically relevant markers Neurochrondrin (EP15001186), ITPR1(EP14003703.7), NBC1 (EP14003958.7), ATP1A3, also referred to as alpha 3subunit of human neuronal Na(+)/K(+) ATPase (EP14171561.5), Flotillin1/2(EP3101424) and RGS8 (EP17000666.2), autoantibodies to one or more ofwhich may be detected in addition, have been described in the state ofthe art.

According to the teachings of the present invention, an antibody,preferably an autoantibody binding to the inventive polypeptide used forthe diagnosis of a disease is provided. The person skilled in the art isfamiliar with methods for purifying antibodies, for example thosedescribed in Hermanson, G. T., Mallia, A. K., and Smith, P. K. (1992),Immobilized Affinity Ligand Techniques, San Diego: Academic Press.Briefly, an antigen binding specifically to the antibody of interest,which antigen is the inventive polypeptide, is immobilized and used topurify, via affinity chromatography, the antibody of interest from anadequate source. A liquid sample comprising antibodies from a patientsuffering from the disease may be used as the source.

According to the invention, an antibody, for example an autoantibody, isprovided that is capable of binding specifically to the inventivepolypeptide. In a preferred embodiment, the term “antibody”, as usedherein, refers to any immunoglobulin-based binding moieties, morepreferably one comprising at least one immunoglobulin heavy chain andone immunoglobulin light chain, including, but not limited to monoclonaland polyclonal antibodies as well as variants of an antibody, inparticular fragments, which binding moieties are capable of binding tothe respective antigen, more preferably binding specifically to it. In apreferred embodiment, the term “binding specifically”, as used herein,means that the binding is stronger than a binding reaction characterizedby a dissociation constant of 1×10⁻⁵ M, more preferably 1×10⁻⁷ M, morepreferably 1×10⁻⁸ M, more preferably 1×10⁻⁹ M, more preferably 1×10⁻¹⁰M, more preferably 1×10⁻¹¹ M, more preferably 1×10⁻¹² M, as determinedby surface plasmon resonance using Biacore equipment at 25° C. in PBSbuffer at pH 7. The antibody may be part of an autoantibody preparationwhich is heterogeneous or may be a homogenous autoantibody, wherein aheterogeneous preparation comprises a plurality of differentautoantibody species as obtainable by preparation from the sera of humandonors, for example by affinity chromatography using the immobilizedantigen to purify any autoantibody capable of binding to said antigen.The antibody may be glycosylated or non-glycosylated. The person skilledin the art is familiar with methods that may be used for theidentification, production and purification of antibodies and variantsthereof, for examples those described in EP 2 423 226 A2 and referencestherein. The antibody may be used as a diagnostic agent, by itself, orin combination, for example in complex with the inventive polypeptide.

The present invention provides a method for isolating an antibody,preferably an autoantibody, binding to the inventive polypeptide,comprising the steps a) contacting a sample comprising the antibody withthe inventive polypeptide such that a complex is formed, b) isolatingthe complex formed in step a), c) dissociating the complex isolated instep b), and d) separating the antibody from the inventive polypeptide.A sample from a patient suffering from the novel neurological disorderidentified by the inventors may be used as the source of antibody.Suitable methods are described in the state of the art, for example inthe Handbooks “Affinity chromatography”, “Strategies for ProteinPurification” and “Antibody Purification” (2009/2010), published by GEHealthcare Life Sciences, and in in Philips, Terry, M., Analyticaltechniques in immunochemistry, 1992, Marcel Dekker, Inc.

The invention provides a pharmaceutical composition comprising theinventive polypeptide, which composition is preferably suitable foradministration to a subject, preferably a mammalian subject, morepreferably to a human. Such a pharmaceutical composition may comprise apharmaceutically acceptable carrier. The pharmaceutical composition may,for example, be administered orally, parenterally, by inhalation spray,topically, by eyedrops, rectally, nasally, buccally, vaginally or via animplanted reservoir, wherein the term “parentally”, as used herein,comprises subcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intrasynovial, instrasternal, intrathecal,intralesional and intracranial injection or infusion techniques. Thepharmaceutical composition may be provided in suitable dosage forms, forexample capsules, tablets and aqueous suspensions and solutions,preferably in sterile form. It may be used in a method of treatment of adisease, which method comprises administering an effective amount of theinventive polypeptide to a subject. In a preferred embodiment, theinvention provides a vaccine comprising the inventive polypeptide,optionally comprising an auxiliary agent such as an adjuvants or abuffer, and the use of the inventive polypeptide for the preparation ofa vaccine.

Within the scope of the present invention, a medical or diagnosticdevice comprising, preferably coated with a reagent for detecting theinventive (auto)antibody and/or the inventive polypeptide is provided.Preferably such a medical or diagnostic device comprises the inventivepolypeptide in a form that allows contacting it with an aqueoussolution, more preferably the liquid human sample, in a straightforwardmanner. In particular, the inventive polypeptide comprising may beimmobilized on the surface of a carrier, preferably selected from thegroup comprising glass plates or slides, biochips, microtiter plates,beads, for example magnetic beads, apharesis devices, chromatographycolumns, membranes or the like. Exemplary medical devices include lineblots, microtiter plates, glass slides for microscopy, beads, preferablymagnetic beads, and biochips. In addition to the inventive polypeptide,the medical or diagnostic device may comprise additional polypeptides,for example positive or negative controls such as samples comprising ornot comprising an antibody binding to the polypeptide of interest, orknown other antigens binding to autoantibodies of diagnostic value,particularly those related other diseases associated with one or moreidentical or similar symptoms.

The inventive teachings provide a kit, preferably for diagnosing adisease. Such a kit may comprise instructions detailing how to use thekit and a means for contacting the inventive polypeptide with a bodilyfluid sample from a subject, preferably a human subject, for example aline blot, wherein the inventive polypeptide is immobilized on the lineblot. Furthermore, the kit may comprise a positive control, for examplea batch of autoantibody or recombinant antibody known to bind to thepolypeptide according to the present invention and a negative control,for example a protein having no detectable affinity to the inventivepolypeptide such as bovine serum albumin. Finally, such a kit maycomprise a standard solution of the antibody or antigen for preparing acalibration curve.

In a preferred embodiment, the kit comprises a means for detecting anautoantibody binding to the inventive polypeptide, preferably bydetecting a complex comprising the inventive polypeptide and an antibodybinding to the inventive polypeptide. Such means is preferably an agentthat binds to said complex and modifies the complex or carries a labelsuch that makes the complex detectable. For example, said means may be alabeled antibody binding to said polypeptide, at a binding site otherthan the binding site recognized by the primary antibody or to aconstant region of the primary antibody. Alternatively, said means maybe a secondary antibody binding to the constant region of theautoantibody, preferably a secondary antibody specific for mammalian IgGclass of antibodies. A multitude of methods and means for detecting sucha complex have been described in the state of the art, for example inPhilips, Terry, M., Analytical techniques in immunochemistry, 1992,Marcel Dekker, Inc.

The polypeptides according to the present invention, comprising apolypeptide selected from the group comprising NSF, STX1B, DNM1 andVAMP2, or a variant thereof may be produced or provided in the form of acell comprising and/or expressing a nucleic acid encoding saidpolypeptide. If a nucleic acid comprising a sequence that encodes forthe inventive polypeptide or variant thereof is used, such a nucleicacid may be an unmodified nucleic acid. In a preferred embodiment, thenucleic acid is a nucleic acid that, as such, does not occur in natureand comprises, compared to natural nucleic acid, at least onemodification, for example an isotopic content or chemical modifications,for example a methylation, sequence modification, label or the likeindicative of synthetic origin. In a preferred embodiment, the nucleicacid is a recombinant nucleic acid, and is, in a more preferredembodiment, part of a vector, in which it may be functionally linkedwith a promoter that allows for expression, preferably overexpression ofthe nucleic acid. The person skilled in the art is familiar with avariety of suitable vectors, of which are commercially available, forexample from Origene. For example, a vector encoding for fusionconstructs with a C-terminal GFP may be used. The cell may be aeukaryotic or prokaryotic cell, preferably of eukaryotic cell, such as ayeast cell, and is more preferably a mammalian, more preferably a humancell such as a HEK293 cell. Examples of a mammalian cell include aHEK293, CHO or COS-7 cell. The cell comprising the nucleic acid encodingfor the inventive polypeptide may be a recombinant cell or an isolatedcell wherein the term “isolated” means that the cell is enriched suchthat, compared to the environment of the wild type of said cell, fewercells of other differentiation or species or in fact no such other cellsare present.

The inventive teachings may not only be used for a diagnosis, but alsofor preventing or treating a disease, more specifically a method forpreventing or treating a disease, comprising the steps a) reducing theconcentration of autoantibodies binding to the inventive polypeptide inthe subject's blood and/or b) administering one or moreimmunosuppressive pharmaceutical substances, preferably selected fromthe group comprising rituximab, prednisone, methylprednisolone,cyclophosphamide, mycophenolatemofetil, intravenous immunoglobulin,tacrolimus, cyclosporine, methotrexate, azathioprine and/or thepharmaceutical composition.

In a preferred embodiment, the present invention provides a use of ameans for the detection of an autoantibody to a polypeptide selectedfrom the group comprising NSF, STX1B, DNM1 and VAMP2, or of a nucleicacid encoding NSF, STX1B, DNM1 or VAMP2 or the variant or a vector orcell comprising said nucleic acid for the manufacture of kit for thediagnosis of a disease such as stiff-person syndrome. In anotherpreferred embodiment, the present invention provides a use of a reagentfor the detection of an autoantibody to a polypeptide selected from thegroup comprising NSF, STX1B, DNM1 and VAMP2, or of a nucleic acidencoding NSF, STX1B, DNM1 or VAMP2 or the variant or a vector or cellcomprising said nucleic acid for the manufacture of kit for thediagnosis of a disease such as stiff-person syndrome.

In a preferred embodiment, any method or use according to the presentinvention may be intended for a non-diagnostic use, i.e. determining thepresence of an autoantibody to a polypeptide selected from the groupcomprising NSF, STX1B, DNM1 and VAMP2 for a use other than diagnosing apatient. For example, the method or use may be for testing in vitro theefficiency of a medical device designed to remove an autoantibody from apatient's blood, wherein the testing is performed on a liquid other thanpatient's blood. In a preferred embodiment, any method or use accordingto the present invention may be intended for generating an autoantibodyprofile, preferably for detecting a disease in a mammal, preferably ahuman. In a preferred embodiment, any method or use may be for detectingdisease-associated markers in a sample from neurological diseasepatients.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows immunofluorescence staining of cerebellum. Cryosectionswere incubated with patient sera (P1 1:32; P2 1:100) in the first step,and with Alexa488-labelled goat anti-human IgG in the second step. Asmooth staining of cerebellar molecular layer and granular layer wasobtained with the strongest reaction on the molecular layer.

FIGS. 2A-2B shows Immunoprecipitation and antigen identification.Lysates of rat cerebellum were incubated with patient or control sera.Immunocomplexes were isolated with protein-G-coated magnetic beads,eluted by SDS and subjected to SDS-PAGE analysis followed by (FIG. 2A)staining with colloidal coomassie or (FIG. 2B) Western blot andincubation with anti-STX1B mouse antibody. Arrow indicates the positionof the immunoprecipitated antigen at about 33 kDa.

FIGS. 3A-3C shows the verification of STX1B as the novel autoantigen byindirect immunofluorescence and Western blot with the recombinantantigen. (FIG. 3A) Indirect immunofluorescence using acetone-fixed STX1Bor mock-transfected HEK293 cells incubated with patient CSF (1:1) orsera (1:10) or a healthy control serum (1:10). (FIG. 3B) Western blotwith STX1B(ic)-His incubated with anti-His, patient sera (1:200) orhealthy control sera (1:200). (FIG. 3C) Neutralization ofimmunofluorescence reaction on neuronal tissues. Patient serum waspre-incubated with extracts of HEK293 cells transfected with STX1B orwith empty vector as control. The extract containing STX1B abolished theimmune reaction.

FIGS. 4A-4C shows the determination of anti-STX1B by IFA and WB.Indirect immunofluorescence using (FIG. 4A) cryosections incubated withpatient sera (1:32) or (FIG. 4B) acetone-fixed STX1B or mock-transfectedHEK293 cells incubated with patient serum (1:10) in the first step, andwith Alexa488-labelled goat anti-human IgG in the second step. (FIG. 4C)Western blot with STX1B(ic)-His incubated with patient sera (1:200) orhealthy control sera (1:200).

FIG. 5 shows the verification of NSF as the novel autoantigen by Westernblot with the recombinant antigen. Western blot with NSF transfectedHEK293 cell extract incubated with anti-His, patient sera or healthycontrol sera (1:1000).

FIGS. 6A-6B shows the verification of VAMP2 as the novel autoantigen byindirect immunofluorescence with the recombinant antigen. Indirectimmunofluorescence using (FIG. 6A) cryosections incubated with patientserum (1:32) or (FIG. 6B) acetone-fixed VAMP2 or mock-transfected HEK293cells incubated with patient or a healthy control serum (1:10) in thefirst step, and with Alexa488-labelled goat anti-human IgG in the secondstep.

FIG. 7 shows an image of a blue silver stained gel following totallysate immunoprecipitation to demonstrate pull-down of DNM1 by patients'sera.

FIG. 8 shows an image of a blue silver stained gel to show pull-down ofDNM1 following cryo-immunoprecipitation.

FIG. 9 shows that patients' sera demonstrate reactivity against DNM1enriched from cerebellum.

FIG. 10 shows that a patient cohort portrayed a significantly higherprevalence of AAbs against DNM1 compared with controls.

The present invention comprises a range of sequences, more specifically

(NSF, UNIPROT) SEQ ID NO: 1MAGRSMQAARCPTDELSLTNCAVVNEKDFQSGQHVIVRTSPNHRYTFTLKTHPSVVPGSIAFSLPQRKWAGLSIGQEIEVSLYTFDKAKQCIGTMTIEIDFLQKKSIDSNPYDTDKMAAEFIQQFNNQAFSVGQQLVFSFNEKLFGLLVKDIEAMDPSILKGEPATGKRQKIEVGLVVGNSQVAFEKAENSSLNLIGKAKTKENRQSIINPDWNFEKMGIGGLDKEFSDIFRRAFASRVFPPEIVEQMGCKHVKGILLYGPPGCGKTLLARQIGKMLNAREPKVVNGPEILNKYVGESEANIRKLFADAEEEQRRLGANSGLHIIIFDEIDAICKQRGSMAGSTGVHDTVVNQLLSKIDGVEQLNNILVIGMTNRPDLIDEALLRPGRLEVKMEIGLPDEKGRLQILHIHTARMRGHQLLSADVDIKELAVETKNFSGAELEGLVRAAQSTAMNRHIKASTKVEVDMEKAESLQVTRGDFLASLENDIKPAFGTNQEDYASYIMNGIIKWGDPVTRVLDDGELLVQQTKNSDRTPLVSVLLEGPPHSGKTALAAKIAEESNFPFIKICSPDKMIGFSETAKCQAMKKIFDDAYKSQLSCVVVDDIERLLDYVPIGPRFSNLVLQALLVLLKKAPPQGRKLLIIGTTSRKDVLQEMEMLNAFSTTIHVPNIATGEQLLEALELLGNFKDKERTTIAQQVKGKKVWIGIKKLLMLIEMSLQMDPEYRVRKFLALLREEGASPLDFD (NSF, REC) SEQ ID NO: 2MAGRSMQAARCPTDELSLTNCAVVNEKDFQSGQHVIVRTSPNHRYTFTLKTHPSVVPGSIAFSLPQRKWAGLSIGQEIEVSLYTFDKAKQCIGTMTIEIDFLQKKSIDSNPYDTDKMAAEFIQQFNNQAFSVGQQLVFSFNEKLFGLLVKDIEAMDPSILKGEPATGKRQKIEVGLVVGNSQVAFEKAENSSLNLIGKAKTKENRQSIINPDWNFEKMGIGGLDKEFSDIFRRAFASRVFPPEIVEQMGCKHVKGILLYGPPGCGKTLLARQIGKMLNAREPKVVNGPEILNKYVGESEANIRKLFADAEEEQRRLGANSGLHIIIFDEIDAICKQRGSMAGSTGVHDTVVNQLLSKIDGVEQLNNILVIGMTNRPDLIDEALLRPGRLEVKMEIGLPDEKGRLQILHIHTARMRGHQLLSADVDIKELAVETKNFSGAELEGLVRAAQSTAMNRHIKASTKVEVDMEKAESLQVTRGDFLASLENDIKPAFGTNQEDYASYIMNGIIKWGDPVTRVLDDGELLVQQTKNSDRTPLVSVLLEGPPHSGKTALAAKIAEESNFPFIKICSPDKMIGFSETAKCQAMKKIFDDAYKSQLSCVVVDDIERLLDYVPIGPRFSNLVLQALLVLLKKAPPQGRKLLIIGTTSRKDVLQEMEMLNAFSTTIHVPNIATGEQLLEALELLGNFKDKERTTIAQQVKGKKVWIGIKKLLMLIEMSLQMDPEYRVRKFLALLREEGASPLDFD (STX1B, UNIPROT) SEQ ID NO: 3MKDRTQELRSAKDSDDEEEVVHVDRDHFMDEFFEQVEEIRGCIEKLSEDVEQVKKQHSAILAAPNPDEKTKQELEDLTADIKKTANKVRSKLKAIEQSIEQEEGLNRSSADLRIRKTQHSTLSRKFVEVMTEYNATQSKYRDRCKDRIQRQLEITGRTTTNEELEDMLESGKLAIFTDDIKMDSQMTKQALNEIETRHNEIIKLETSIRELHDMFVDMAMLVESQGEMIDRIEYNVEHSVDYVERAVSDTKKAVKYQSKARRKKIMIIICCVVLGVVLASSIGGTLGL (STX1B, REC) SEQ ID NO: 4MKDRTQELRSAKDSDDEEEVVHVDRDHFMDEFFEQVEEIRGCIEKLSEDVEQVKKQHSAILAAPNPDEKTKQELEDLTADIKKTANKVRSKLKAIEQSIEQEEGLNRSSADLRIRKTQHSTLSRKFVEVMTEYNATQSKYRDRCKDRIQRQLEITGRTTTNEELEDMLESGKLAIFTDDIKMDSQMTKQALNEIETRHNEIIKLETSIRELHDMFVDMAMLVESQGEMIDRIEYNVEHSVDYVERAVSDTKKAVKYQSKARRKKIMIIICCVVLGVVLASSIGGTLGL (STX1B(ic)-His, REC) SEQ ID NO: 5MKDRTQELRSAKDSDDEEEVVHVDRDHFMDEFFEQVEEIRGCIEKLSEDVEQVKKQHSAILAAPNPDEKTKQELEDLTADIKKTANKVRSKLKAIEQSIEQEEGLNRSSADLRIRKTQHSTLSRKFVEVMTEYNATQSKYRDRCKDRIQRQLEITGRTTTNEELEDMLESGKLAIFTDDIKMDSQMTKQALNEIETRHNEIIKLETSIRELHDMFVDMAMLVESQGEMIDRIEYNVEHSVDYVERAVSDTKKAVKYQSKARRKKLEHHHHHHHH (VAMP2, UNIPROT) SEQ ID NO: 6MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST (VAMP2, REC)SEQ ID NO: 7MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST (sense NSF)SEQ ID NO: 8 ATACGTCTCACATGGCGGGCCGGAGCATGCAAG (asense NSF) SEQ ID NO: 9TATCGTCTCCTCGATCAATCAAAATCAAGGGGGCTAG (sense STX1B) SEQ ID NO: 10ATACGTCTCACATGAAGGATCGGACTCAAGAGCTGC (asense STX1B) SEQ ID NO: 11ATACGTCTCCTCGAGCTACAAGCCCAGCGTCCCCCCAATG (asense STX1B(ic)-His)SEQ ID NO: 12 ATACGTCTCCTCGAGTTTCTTCCTCCGGGCCTTGCTCTG (sense VAMP2)SEQ ID NO: 13 ATACGTCTCTCATGTCTGCTACCGCTGCCACGGCCC (asense VAMP2)SEQ ID NO: 14 ATACGTCTCCTCGAGTTAAGTGCTGAAGTAAACTATGATG (pTriEx-1-NSF)SEQ ID NO: 15AATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCGGAGTTAATCCGGGACCTTTAATTCAACCCAACACAATATATTATAGTTAAATAAGAATTATTATCAAATCATTTGTATATTAATTAAAATACTATACTGTAAATTACATTTTATTTACAATCAAAGGAGATATACCATGGCGGGCCGGAGCATGCAAGCGGCAAGATGTCCTACAGATGAATTATCTTTAACCAATTGTGCAGTTGTGAATGAAAAGGATTTCCAGTCTGGCCAGCATGTGATTGTGAGGACCTCTCCCAATCACAGGTACACATTTACACTGAAGACACATCCATCGGTGGTTCCAGGGAGCATTGCATTCAGTTTACCTCAGAGAAAATGGGCTGGGCTTTCTATTGGGCAAGAAATAGAAGTCTCCTTATATACATTTGACAAAGCCAAACAGTGTATTGGCACAATGACCATCGAGATTGATTTCCTGCAGAAAAAAAGCATTGACTCCAACCCTTATGACACCGACAAGATGGCAGCAGAATTTATTCAGCAATTCAACAACCAGGCCTTCTCAGTGGGACAACAGCTTGTCTTTAGCTTCAATGAAAAGCTTTTTGGCTTACTGGTGAAGGACATTGAAGCCATGGATCCTAGCATCCTGAAGGGAGAGCCTGCGACAGGGAAAAGGCAGAAGATTGAAGTAGGACTGGTTGTTGGAAACAGTCAAGTTGCATTTGAAAAAGCAGAAAATTCGTCACTTAATCTTATTGGCAAAGCTAAAACCAAGGAAAATCGCCAATCAATTATCAATCCTGACTGGAACTTTGAAAAAATGGGAATAGGAGGTCTAGACAAGGAATTTTCAGATATTTTCCGACGAGCATTTGCTTCCCGAGTATTTCCTCCAGAGATTGTGGAGCAGATGGGTTGTAAACATGTTAAAGGCATCCTGTTATATGGACCCCCAGGTTGTGGTAAGACTCTCTTGGCTCGACAGATTGGCAAGATGTTGAATGCAAGAGAGCCCAAAGTGGTCAATGGGCCAGAAATCCTTAACAAATATGTGGGAGAATCAGAGGCTAACATTCGCAAACTTTTTGCTGATGCTGAAGAGGAGCAAAGGAGGCTTGGTGCTAACAGTGGTTTGCACATCATCATCTTTGATGAAATTGATGCCATCTGCAAGCAGAGAGGGAGCATGGCTGGTAGCACGGGAGTTCATGACACTGTTGTCAACCAGTTGCTGTCCAAAATTGATGGCGTGGAGCAGCTAAACAACATCCTAGTCATTGGAATGACCAATAGACCAGATCTGATAGATGAGGCTCTTCTTAGACCTGGAAGACTGGAAGTTAAAATGGAGATAGGCTTGCCAGATGAGAAAGGCCGACTACAGATTCTTCACATCCACACAGCAAGAATGAGAGGGCATCAGTTACTCTCTGCTGATGTAGACATTAAAGAACTGGCCGTGGAGACCAAGAATTTCAGTGGTGCTGAATTGGAGGGTCTAGTGCGAGCAGCCCAGTCCACTGCTATGAATAGACACATAAAGGCCAGTACTAAAGTGGAAGTGGACATGGAGAAAGCAGAAAGCCTGCAAGTGACGAGAGGAGACTTCCTTGCTTCTTTGGAGAATGATATCAAACCAGCCTTTGGCACAAACCAAGAAGATTATGCAAGTTACATTATGAACGGTATCATCAAATGGGGTGACCCAGTTACTCGAGTTCTAGATGATGGGGAGCTGCTGGTGCAGCAGACTAAGAACAGTGACCGCACACCATTGGTCAGCGTGCTTCTGGAAGGCCCTCCTCACAGTGGGAAGACTGCTTTAGCTGCAAAAATTGCAGAGGAATCCAACTTCCCATTCATCAAGATCTGTTCTCCTGATAAAATGATTGGCTTTTCTGAAACAGCCAAATGTCAGGCCATGAAGAAGATCTTTGATGATGCGTACAAATCCCAGCTCAGTTGTGTGGTTGTGGATGACATTGAGAGATTGCTTGATTACGTCCCTATTGGCCCTCGATTTTCAAATCTTGTATTACAGGCTCTTCTCGTTTTACTGAAAAAGGCACCTCCTCAGGGCCGCAAGCTTCTTATCATTGGGACCACTAGCCGCAAAGATGTCCTTCAGGAGATGGAAATGCTTAACGCTTTCAGCACCACCATCCACGTGCCCAACATTGCCACAGGAGAGCAGCTGTTGGAAGCTTTGGAGCTTTTGGGCAACTTCAAGGATAAGGAACGCACCACAATTGCACAGCAAGTCAAAGGGAAGAAGGTCTGGATAGGAATCAAGAAGTTACTAATGCTGATCGAGATGTCCCTACAGATGGATCCTGAATACCGTGTGAGAAAATTCTTGGCCCTCTTAAGAGAAGAAGGAGCTAGCCCCCTTGATTTTGATTGATCGAGCACCACCATCACCATCACCATCACTAAGTGATTAACCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGTAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGCATGCGGAGGAAATTCTCCTTGAAGTTTCCCTGGTGTTCAAAGTAAAGGAGTTTGCACCAGACGCACCTCTGTTCACTGGTCCGGCGTATTAAAACACGATACATTGTTATTAGTACATTTATTAAGCGCTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTACGTATTTTAATAATTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGCGAAAATCAAATGATTTTCAGCGTCTTTATATCTGAATTTAAATATTAAATCCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCAAACAAGGGTTGTTTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAACGCCACAAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTTGTGTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCTTTTGTATTTCTTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAACACGTTAAATAGAGCTTGGACATATTTAACATCGGGCGTGTTAGCTTTATTAGGCCGATTATCGTCGTCGTCCCAACCCTCGTCGTTAGAAGTTGCTTCCGAAGACGATTTTGCCATAGCCACACGACGCCTATTAATTGTGTCGGCTAACACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAATTATTTCTGATTGCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAATTCAGACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGGCAAATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCGGTGGAGGCGCAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGCGGTGATGCAGACGGCGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGTCGGCACCTCAACTATTGTACTGGTTTCGGGCGCCGTTTTTGGTTTGACCGGTCTGAGACGAGTGCGATTTTTTTCGTTTCTAATAGCTTCCAACAATTGTTGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCCGTCGGCATTGGTGGAGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGGAGGCGCTGGAATGTTAGGCACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTATAATTTGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGCCGCTGGCTGCACAACGGAAGGTCGTCTGCTTCGAGGCAGCGCTTGGGGTGGTGGCAATTCAATATTATAATTGGAATACAAATCGTAAAAATCTGCTATAAGCATTGTAATTTCGCTATCGTTTACCGTGCCGATATTTAACAACCGCTCAATGTAAGCAATTGTATTGTAAAGAGATTGTCTCAAGCTCGGAACGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGTCCGCGCGTTTCCTGCATCTTTTAATCAAATCCCAAGATGTGTATAAACCACCAAACTGCCAAAAAATGAAAACTGTCGACAAGCTCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTATTGAATAATAAAACAATTATAAATGTCAAATTTGTTTTTTATTAACGATACAAACCAAACGCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGCGTAGTTATAATCGCTGAGGTAATATTTAAAATCATTTTCAAATGATTCACAGTTAATTTGCGACAATATAATTTTATTTTCACATAAACTAGACGCCTTGTCGTCTTCTTCTTCGTATTCCTTCTCTTTTTCATTTTTCTCTTCATAAAAATTAACATAGTTATTATCGTATCCATATATGTATCTATCGTATAGAGTAAATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAGTACCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAGTTCTTCGGAGTGTGTTGCTTTAATTATTAAATTTATATAATCAATGAATTTGGGATCGTCGGTTTTGTACAATATGTTGCCGGCATAGTACGCAGCTTCTTCTAGTTCAATTACACCATTTTTTAGCAGCACCGGATTAACATAACTTTCCAAAATGTTGTACGAACCGTTAAACAAAAACAGTTCACCTCCCTTTTCTATACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAATAACAGCCATTGTAATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCAATATATAGTTGCTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTTCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCGGACCGAAAT (pTriEx-1-STX1B) SEQ ID NO: 16GGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTTCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCGGACCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCGGAGTTAATCCGGGACCTTTAATTCAACCCAACACAATATATTATAGTTAAATAAGAATTATTATCAAATCATTTGTATATTAATTAAAATACTATACTGTAAATTACATTTTATTTACAATCAAAGGAGATATACCATGAAGGATCGGACTCAAGAGCTGCGGAGTGCGAAAGACAGTGATGATGAAGAGGAGGTGGTCCACGTGGATCGGGACCACTTCATGGATGAGTTCTTTGAACAGGTGGAAGAGATCCGGGGCTGCATTGAGAAACTGTCGGAGGATGTGGAGCAGGTGAAAAAACAGCATAGCGCCATCCTGGCCGCACCCAACCCAGATGAGAAGACCAAACAGGAGCTGGAGGATCTCACTGCAGACATCAAGAAGACGGCCAACAAGGTTCGGTCCAAATTGAAAGCGATCGAGCAAAGCATTGAACAGGAGGAGGGGCTGAACCGTTCCTCCGCGGACCTGCGCATCCGCAAGACCCAGCACTCCACACTGTCCCGGAAGTTCGTGGAGGTAATGACCGAATATAACGCGACCCAGTCCAAGTACCGGGACCGCTGCAAGGACCGGATCCAGCGGCAACTGGAGATCACTGGAAGGACCACCACCAACGAAGAACTGGAAGACATGCTGGAGAGCGGGAAGCTGGCCATCTTCACAGATGACATCAAAATGGACTCACAGATGACGAAGCAGGCGCTGAATGAGATTGAGACGAGGCACAATGAGATCATCAAGCTGGAGACCAGCATCCGCGAGCTGCACGATATGTTTGTGGACATGGCCATGCTCGTAGAGAGCCAGGGAGAGATGATTGACCGCATCGAGTACAACGTGGAACATTCTGTGGACTACGTGGAGCGAGCTGTGTCTGACACCAAGAAAGCAGTGAAATATCAGAGCAAGGCCCGGAGGAAGAAAATCATGATCATCATTTGCTGTGTGGTGCTGGGGGTGGTCTTGGCGTCGTCCATTGGGGGGACGCTGGGCTTGTAGCTCGAGCACCACCATCACCATCACCATCACTAAGTGATTAACCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGTAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGCATGCGGAGGAAATTCTCCTTGAAGTTTCCCTGGTGTTCAAAGTAAAGGAGTTTGCACCAGACGCACCTCTGTTCACTGGTCCGGCGTATTAAAACACGATACATTGTTATTAGTACATTTATTAAGCGCTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTACGTATTTTAATAATTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGCGAAAATCAAATGATTTTCAGCGTCTTTATATCTGAATTTAAATATTAAATCCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCAAACAAGGGTTGTTTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAACGCCACAAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTTGTGTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCTTTTGTATTTCTTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAACACGTTAAATAGAGCTTGGACATATTTAACATCGGGCGTGTTAGCTTTATTAGGCCGATTATCGTCGTCGTCCCAACCCTCGTCGTTAGAAGTTGCTTCCGAAGACGATTTTGCCATAGCCACACGACGCCTATTAATTGTGTCGGCTAACACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAATTATTTCTGATTGCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAATTCAGACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGGCAAATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCGGTGGAGGCGCAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGCGGTGATGCAGACGGCGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGTCGGCACCTCAACTATTGTACTGGTTTCGGGCGCCGTTTTTGGTTTGACCGGTCTGAGACGAGTGCGATTTTTTTCGTTTCTAATAGCTTCCAACAATTGTTGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCCGTCGGCATTGGTGGAGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGGAGGCGCTGGAATGTTAGGCACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTATAATTTGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGCCGCTGGCTGCACAACGGAAGGTCGTCTGCTTCGAGGCAGCGCTTGGGGTGGTGGCAATTCAATATTATAATTGGAATACAAATCGTAAAAATCTGCTATAAGCATTGTAATTTCGCTATCGTTTACCGTGCCGATATTTAACAACCGCTCAATGTAAGCAATTGTATTGTAAAGAGATTGTCTCAAGCTCGGAACGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGTCCGCGCGTTTCCTGCATCTTTTAATCAAATCCCAAGATGTGTATAAACCACCAAACTGCCAAAAAATGAAAACTGTCGACAAGCTCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTATTGAATAATAAAACAATTATAAATGTCAAATTTGTTTTTTATTAACGATACAAACCAAACGCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGCGTAGTTATAATCGCTGAGGTAATATTTAAAATCATTTTCAAATGATTCACAGTTAATTTGCGACAATATAATTTTATTTTCACATAAACTAGACGCCTTGTCGTCTTCTTCTTCGTATTCCTTCTCTTTTTCATTTTTCTCTTCATAAAAATTAACATAGTTATTATCGTATCCATATATGTATCTATCGTATAGAGTAAATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAGTACCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAGTTCTTCGGAGTGTGTTGCTTTAATTATTAAATTTATATAATCAATGAATTTGGGATCGTCGGTTTTGTACAATATGTTGCCGGCATAGTACGCAGCTTCTTCTAGTTCAATTACACCATTTTTTAGCAGCACCGGATTAACATAACTTTCCAAAATGTTGTACGAACCGTTAAACAAAAACAGTTCACCTCCCTTTTCTATACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAATAACAGCCATTGTAATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCAATATATAGTTGCTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG (pTriEx-1-STX1B(ic)-His) SEQ ID NO: 17GGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTTCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCGGACCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCGGAGTTAATCCGGGACCTTTAATTCAACCCAACACAATATATTATAGTTAAATAAGAATTATTATCAAATCATTTGTATATTAATTAAAATACTATACTGTAAATTACATTTTATTTACAATCAAAGGAGATATACCATGAAGGATCGGACTCAAGAGCTGCGGAGTGCGAAAGACAGTGATGATGAAGAGGAGGTGGTCCACGTGGATCGGGACCACTTCATGGATGAGTTCTTTGAACAGGTGGAAGAGATCCGGGGCTGCATTGAGAAACTGTCGGAGGATGTGGAGCAGGTGAAAAAACAGCATAGCGCCATCCTGGCCGCACCCAACCCAGATGAGAAGACCAAACAGGAGCTGGAGGATCTCACTGCAGACATCAAGAAGACGGCCAACAAGGTTCGGTCCAAATTGAAAGCGATCGAGCAAAGCATTGAACAGGAGGAGGGGCTGAACCGTTCCTCCGCGGACCTGCGCATCCGCAAGACCCAGCACTCCACACTGTCCCGGAAGTTCGTGGAGGTAATGACCGAATATAACGCGACCCAGTCCAAGTACCGGGACCGCTGCAAGGACCGGATCCAGCGGCAACTGGAGATCACTGGAAGGACCACCACCAACGAAGAACTGGAAGACATGCTGGAGAGCGGGAAGCTGGCCATCTTCACAGATGACATCAAAATGGACTCACAGATGACGAAGCAGGCGCTGAATGAGATTGAGACGAGGCACAATGAGATCATCAAGCTGGAGACCAGCATCCGCGAGCTGCACGATATGTTTGTGGACATGGCCATGCTCGTAGAGAGCCAGGGAGAGATGATTGACCGCATCGAGTACAACGTGGAACATTCTGTGGACTACGTGGAGCGAGCTGTGTCTGACACCAAGAAAGCAGTGAAATATCAGAGCAAGGCCCGGAGGAAGAAACTCGAGCACCACCATCACCATCACCATCACTAAGTGATTAACCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGTAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGCATGCGGAGGAAATTCTCCTTGAAGTTTCCCTGGTGTTCAAAGTAAAGGAGTTTGCACCAGACGCACCTCTGTTCACTGGTCCGGCGTATTAAAACACGATACATTGTTATTAGTACATTTATTAAGCGCTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTACGTATTTTAATAATTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGCGAAAATCAAATGATTTTCAGCGTCTTTATATCTGAATTTAAATATTAAATCCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCAAACAAGGGTTGTTTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAACGCCACAAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTTGTGTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCTTTTGTATTTCTTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAACACGTTAAATAGAGCTTGGACATATTTAACATCGGGCGTGTTAGCTTTATTAGGCCGATTATCGTCGTCGTCCCAACCCTCGTCGTTAGAAGTTGCTTCCGAAGACGATTTTGCCATAGCCACACGACGCCTATTAATTGTGTCGGCTAACACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAATTATTTCTGATTGCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAATTCAGACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGGCAAATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCGGTGGAGGCGCAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGCGGTGATGCAGACGGCGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGTCGGCACCTCAACTATTGTACTGGTTTCGGGCGCCGTTTTTGGTTTGACCGGTCTGAGACGAGTGCGATTTTTTTCGTTTCTAATAGCTTCCAACAATTGTTGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCCGTCGGCATTGGTGGAGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGGAGGCGCTGGAATGTTAGGCACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTATAATTTGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGCCGCTGGCTGCACAACGGAAGGTCGTCTGCTTCGAGGCAGCGCTTGGGGTGGTGGCAATTCAATATTATAATTGGAATACAAATCGTAAAAATCTGCTATAAGCATTGTAATTTCGCTATCGTTTACCGTGCCGATATTTAACAACCGCTCAATGTAAGCAATTGTATTGTAAAGAGATTGTCTCAAGCTCGGAACGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGTCCGCGCGTTTCCTGCATCTTTTAATCAAATCCCAAGATGTGTATAAACCACCAAACTGCCAAAAAATGAAAACTGTCGACAAGCTCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTATTGAATAATAAAACAATTATAAATGTCAAATTTGTTTTTTATTAACGATACAAACCAAACGCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGCGTAGTTATAATCGCTGAGGTAATATTTAAAATCATTTTCAAATGATTCACAGTTAATTTGCGACAATATAATTTTATTTTCACATAAACTAGACGCCTTGTCGTCTTCTTCTTCGTATTCCTTCTCTTTTTCATTTTTCTCTTCATAAAAATTAACATAGTTATTATCGTATCCATATATGTATCTATCGTATAGAGTAAATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAGTACCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAGTTCTTCGGAGTGTGTTGCTTTAATTATTAAATTTATATAATCAATGAATTTGGGATCGTCGGTTTTGTACAATATGTTGCCGGCATAGTACGCAGCTTCTTCTAGTTCAATTACACCATTTTTTAGCAGCACCGGATTAACATAACTTTCCAAAATGTTGTACGAACCGTTAAACAAAAACAGTTCACCTCCCTTTTCTATACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAATAACAGCCATTGTAATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCAATATATAGTTGCTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG (pTriEx-1-VAMP2) SEQ ID NO: 18GGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTTCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCGGACCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCGGAGTTAATCCGGGACCTTTAATTCAACCCAACACAATATATTATAGTTAAATAAGAATTATTATCAAATCATTTGTATATTAATTAAAATACTATACTGTAAATTACATTTTATTTACAATCAAAGGAGATATACCATGTCTGCTACCGCTGCCACGGCCCCCCCTGCTGCCCCGGCTGGGGAGGGTGGTCCCCCTGCACCCCCTCCAAACCTCACCAGTAACAGGAGACTGCAGCAGACCCAGGCCCAGGTGGATGAGGTGGTGGACATCATGAGGGTGAACGTGGACAAGGTCCTGGAGCGAGACCAGAAGCTGTCGGAGCTGGACGACCGTGCAGATGCACTCCAGGCGGGGGCCTCCCAGTTTGAAACAAGCGCAGCCAAGCTCAAGCGCAAATACTGGTGGAAAAACCTCAAGATGATGATCATCTTGGGAGTGATTTGCGCCATCATCCTCATCATCATCATAGTTTACTTCAGCACTTAACTCGAGCACCACCATCACCATCACCATCACTAAGTGATTAACCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGTAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGCATGCGGAGGAAATTCTCCTTGAAGTTTCCCTGGTGTTCAAAGTAAAGGAGTTTGCACCAGACGCACCTCTGTTCACTGGTCCGGCGTATTAAAACACGATACATTGTTATTAGTACATTTATTAAGCGCTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTACGTATTTTAATAATTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGCGAAAATCAAATGATTTTCAGCGTCTTTATATCTGAATTTAAATATTAAATCCTCAATAGATTTGTAAAATAGGTTTCGATTAGTTTCAAACAAGGGTTGTTTTTCCGAACCGATGGCTGGACTATCTAATGGATTTTCGCTCAACGCCACAAAACTTGCCAAATCTTGTAGCAGCAATCTAGCTTTGTCGATATTCGTTTGTGTTTTGTTTTGTAATAAAGGTTCGACGTCGTTCAAAATATTATGCGCTTTTGTATTTCTTTCATCACTGTCGTTAGTGTACAATTGACTCGACGTAAACACGTTAAATAGAGCTTGGACATATTTAACATCGGGCGTGTTAGCTTTATTAGGCCGATTATCGTCGTCGTCCCAACCCTCGTCGTTAGAAGTTGCTTCCGAAGACGATTTTGCCATAGCCACACGACGCCTATTAATTGTGTCGGCTAACACGTCCGCGATCAAATTTGTAGTTGAGCTTTTTGGAATTATTTCTGATTGCGGGCGTTTTTGGGCGGGTTTCAATCTAACTGTGCCCGATTTTAATTCAGACAACACGTTAGAAAGCGATGGTGCAGGCGGTGGTAACATTTCAGACGGCAAATCTACTAATGGCGGCGGTGGTGGAGCTGATGATAAATCTACCATCGGTGGAGGCGCAGGCGGGGCTGGCGGCGGAGGCGGAGGCGGAGGTGGTGGCGGTGATGCAGACGGCGGTTTAGGCTCAAATGTCTCTTTAGGCAACACAGTCGGCACCTCAACTATTGTACTGGTTTCGGGCGCCGTTTTTGGTTTGACCGGTCTGAGACGAGTGCGATTTTTTTCGTTTCTAATAGCTTCCAACAATTGTTGTCTGTCGTCTAAAGGTGCAGCGGGTTGAGGTTCCGTCGGCATTGGTGGAGCGGGCGGCAATTCAGACATCGATGGTGGTGGTGGTGGTGGAGGCGCTGGAATGTTAGGCACGGGAGAAGGTGGTGGCGGCGGTGCCGCCGGTATAATTTGTTCTGGTTTAGTTTGTTCGCGCACGATTGTGGGCACCGGCGCAGGCGCCGCTGGCTGCACAACGGAAGGTCGTCTGCTTCGAGGCAGCGCTTGGGGTGGTGGCAATTCAATATTATAATTGGAATACAAATCGTAAAAATCTGCTATAAGCATTGTAATTTCGCTATCGTTTACCGTGCCGATATTTAACAACCGCTCAATGTAAGCAATTGTATTGTAAAGAGATTGTCTCAAGCTCGGAACGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGTCCGCGCGTTTCCTGCATCTTTTAATCAAATCCCAAGATGTGTATAAACCACCAAACTGCCAAAAAATGAAAACTGTCGACAAGCTCTGTCCGTTTGCTGGCAACTGCAAGGGTCTCAATCCTATTTGTAATTATTGAATAATAAAACAATTATAAATGTCAAATTTGTTTTTTATTAACGATACAAACCAAACGCAACAAGAACATTTGTAGTATTATCTATAATTGAAAACGCGTAGTTATAATCGCTGAGGTAATATTTAAAATCATTTTCAAATGATTCACAGTTAATTTGCGACAATATAATTTTATTTTCACATAAACTAGACGCCTTGTCGTCTTCTTCTTCGTATTCCTTCTCTTTTTCATTTTTCTCTTCATAAAAATTAACATAGTTATTATCGTATCCATATATGTATCTATCGTATAGAGTAAATTTTTTGTTGTCATAAATATATATGTCTTTTTTAATGGGGTGTATAGTACCGCTGCGCATAGTTTTTCTGTAATTTACAACAGTGCTATTTTCTGGTAGTTCTTCGGAGTGTGTTGCTTTAATTATTAAATTTATATAATCAATGAATTTGGGATCGTCGGTTTTGTACAATATGTTGCCGGCATAGTACGCAGCTTCTTCTAGTTCAATTACACCATTTTTTAGCAGCACCGGATTAACATAACTTTCCAAAATGTTGTACGAACCGTTAAACAAAAACAGTTCACCTCCCTTTTCTATACTATTGTCTGCGAGCAGTTGTTTGTTGTTAAAAATAACAGCCATTGTAATGAGACGCACAAACTAATATCACAAACTGGAAATGTCTATCAATATATAGTTGCTCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG (DNM1) SEQ ID NO: 19MGNRGMEDLIPLVNRLQDAFSAIGQNADLDLPQIAVVGGQSAGKSSVLENFVGRDFLPRGSGIVTRRPLVLQLVNATTEYAEFLHCKGKKFTDFEEVRLEIEAETDRVTGTNKGISPVPINLRVYSPHVLNLTLVDLPGMTKVPVGDQPPDIEFQIRDMLMQFVTKENCLILAVSPANSDLANSDALKVAKEVDPQGQRTIGVITKLDLMDEGTDARDVLENKLLPLRRGYIGVVNRSQKDIDGKKDITAALAAERKFFLSHPSYRHLADRMGTPYLQKVLNQQLTNHIRDTLPGLRNKLQSQLLSIEKEVEEYKNFRPDDPARKTKALLQMVQQFAVDFEKRIEGSGDQIDTYELSGGARINRIFHERFPFELVKMEFDEKELRREISYAIKNIHGIRTGLFTPDMAFETIVKKQVKKIREPCLKCVDMVISELISTVRQCTKKLQQYPRLREEMERIVTTHIREREGRTKEQVMLLIDIELAYMNTNHEDFIGFANAQQRSNQMNKKKTSGNQDEILVIRKGWLTINNIGIMKGGSKEYWFVLTAENLSVVYKDDEEKEKKYMLSVDNLKLRDVEKGFMSSKHIFALFNTEQRNVYKDYRQLELACETQEEVDSWKASFLRAGVYPERVGDKEKASETEENGSDSFMHSMDPQLERQVETIRNLVDSYMAIVNKTVRDLMPKTIMHLMINNTKEFIFSELLANLYSCGDQNTLMEESAEQAQRRDEMLRMYHALKEALSIIGDINTTTVSTPMPPPVDDSWLQVQSVPAGRRSPTSSPTPQRRAPAVPPARPGSRGPAPGPPPAGSALGGAPPVPSRPGASPDPFGPPPQVPSRPNRAPPGVPSRSGQASPSRPESPRPPFDL SEQ ID NO: 20gggcgggggccccgcggcgcaggcagtctgggcgcgcggctgcagcggcggagccggagtcggagccgggagcgctagcggcagccggatcgcagcctgcggggcccgccgcagccatgggcaaccgcggcatggaagatctcatcccgctggtcaaccggctgcaagacgccttctctgccatcggccagaacgcggacctcgacctgccgcagatcgctgtggtgggcggccagagcgccggcaagagctcggtgctcgagaatttcgtaggcagggacttcttgcctcgaggatctggcattgtcacccgacgtcccctggtcttgcagctggtcaatgcaaccacagaatatgccgagttcctgcactgcaagggaaagaaattcaccgacttcgaggaggtgcgccttgagatcgaggccgagaccgacagggtcaccggcaccaacaagggcatctcgccggtgcctatcaacctccgcgtctactcgccgcacgtgctgaacctgaccctggtggacctgcccggaatgaccaaggtcccggtgggggaccaacctcccgacatcgagttccagatccgagacatgcttatgcagtttgtcaccaaggagaactgcctcatcctggccgtgtcccccgccaactctgacctggccaattctgacgccctcaaggtcgccaaggaggtggacccccagggccagcgcaccatcggggtcatcaccaagctggacctgatggacgagggcacagatgcccgtgatgtgctggagaacaagctgctccccctgcgcagaggctacattggagtggtgaaccggagccagaaggacattgatggcaagaaggacattaccgccgccttggctgctgaacgaaagttcttcctctcccatccatcttatcgccacttggctgaccgtatgggcacgccctacctgcagaaggtcctcaatcagcaactgacgaaccacatccgggacacactgccggggctgcggaacaagctgcagagccagctactgtccattgagaaggaggtggaggaatacaagaacttccgccctgatgacccagctcgcaagaccaaggccctgctgcagatggtccagcagttcgccgtagactttgagaagcgcattgagggctcaggagatcagatcgacacctacgaactgtcagggggagcccgcattaaccgaatcttccacgagcgcttccctttcgagctggtcaagatggagtttgatgagaaggaactccgaagggagatcagctatgctatcaagaatatccatggcattagaacggggctgtttaccccagacatggcctttgagaccattgtgaaaaagcaggtgaagaagatccgagaaccgtgtctcaagtgtgtggacatggttatctcggagctaatcagcaccgttagacagtgcaccaagaagctccagcagtacccgcggctacgggaggagatggagcgcatcgtgaccacccacatccgggagcgcgagggccgcactaaggagcaggtcatgcttctcatcgatatcgagctggcttacatgaacaccaaccatgaggacttcataggctttgccaatgctcagcagaggagcaaccagatgaacaagaagaagacttcagggaaccaggatgagattctggtcatccgcaagggctggctgactatcaataatattggcatcatgaaagggggctccaaggagtactggthgtgctgactgctgagaatctgtcctggtacaaggatgatgaggagaaagagaagaaatacatgctgtctgtggacaacctcaagctgcgggacgtggagaagggctttatgtcgagcaagcatatctttgccctctttaacacggagcagaggaatgtctacaaggattatcggcagctggagctagcctgtgagacacaggaggaggtggacagctggaaggcctccttcctgagggctggcgtgtaccctgagcgtgttggggacaaagagaaagccagcgagaccgaggagaatggctccgacagcttcatgcattccatggacccacagctggaacggcaagtggagaccatccggaatcttgtggactcatacatggccattgtcaacaagaccgtgagggacctcatgcccaagaccatcatgcacctcatgattaacaataccaaggagttcatcttctcggagctgctggccaacctgtactcgtgtggggaccagaacacgctgatggaggagtcggcggagcaggcacagcggcgcgacgagatgctgcgcatgtaccacgcactgaaggaggcgctcagcatcatcggcgacatcaacacgaccaccgtcagcacgcccatgcccccgcccgtggacgactcctggctgcaggtgcagagcgtaccggccggacgcaggtcgcccacgtccagccccacgccgcagcgccgagcccccgccgtgcccccagcccggcccgggtcgcggggccctgctcctgggcctccgcctgctgggtccgccctggggggggcgccccccgtgccctccaggccgggggcttcccctgaccctttcggccctccccctcaggtgccctcgcgccccaaccgcgccccgcccggggtccccagccgatcgggtcaggcaagtccatcccgtcctgagagccccaggccccccttcgacctctaaacagatccctcctcttctcggagacctccctttccaagcctgcctggacggctgttctgtgacttgacagtggctcccccagccccaaagccagcccccttcatctgtgacttaatctgttgtagtggtgagctgatacattcaggtgtgaccgttggtgaaaacttgtgccccttctgtggtatgcccttgccctgttctataaatatctataaatactcatatatatacacacctacacatggccaaccgcctcgcctctagcgctgggaatcagtcactgtgctatccttgtggagtcttgtggcccaactaccagagaacgctgtcccccgacatcccactccaaagtgtgccacctccagtgagcctccttgtcatgcccggcctgtggacagccagcccccgccatccctcccaccccctaccaagcatgggggtgctgtgcaggcagccgtgtggcctgacagtttctaccagtcctgctgtccctcggctgagaataaaacccatttctggatgatggggaatgtcaaaaaaaaaaaaaaa

The present invention is further illustrated by the followingnon-limiting examples from which further features, embodiments, aspectsand advantages of the present invention may be taken.

EXAMPLES

Summary

Methods:

Two patients (P1-P2) with idiopathic encephalitis and an autoimmunebackground underwent serological investigation. For this purpose, serafrom both patients and matched cerebrospinal fluid (CSF) from P2 weresubjected to comprehensive autoantibody screening by indirectimmunofluorescence assay (IFA) and immunoblot. Immunoprecipitation withlysates of cerebellum followed by mass spectrometry (MS) was used toidentify the autoantigen, which was verified by Western blot (WB) withmonospecifc animal antibody against the respective target antigen aswell as by recombinant expression in HEK293 cells and use of therecombinant protein in immunoassays. Furthermore, sera of patients withneurological symptoms and defined anti-neural autoantibodies, sera witha similar staining pattern as patient 1 and 2 without known autoantibodyreactivity, as well as negative control sera were screened foranti-STX1B antibodies. All sera were additionally analyzed by IFA orWestern blot with other recombinant SNARE complex proteins (VAMP2, NSF)as substrates.

Results:

IFA screening of P1 and P2 revealed IgG reactivity in sera and CSF withthe molecular and granular layers in rodent and monkey cerebellum.Furthermore, no IgG reactivity was found with a panel of 30recombinantly expressed established neural autoantigens. The sera of P1and P2 immunoprecipitated syntaxin 1B (STX1B), as detected byCoomassie-stained SDS-PAGE followed by MALDI-TOF mass spectrometry. Whenthe immunoprecipitates were analyzed by Western blot using monospecifcanimal antibodies against STX1B, anti-STX1B showed reactivity with theimmunoprecipitate of P1 and P2. Anti-STX1B antibodies were not found inany of 45 healthy controls. However, in two patient sera (P3 and P4)with a similar staining pattern on cerebellum as P1 and P2 anti-STX1Bantibodies could be detected by RC-IFA and Western blot with therecombinant protein. Furthermore, anti-GAD65 positive sera of twopatients who were pre-diagnosed with stiff person syndrome (P6 and P7)were positive in IFA with recombinant STX1B. Screening of control andanti-STX1B positive sera against other recombinant SNARE proteinsrevealed three anti-NSF (P3, P6 and P7) positive and one anti-VAMP2 (P5)positive sample.

These results show that the emergence and detection of an autoantibodyis specifically linked to the emergence of AE and SPS, respectively,and, consequently, diagnostically useful.

Patients

Control collectives included 45 healthy donors, 33 patients withneurological symptoms and defined anti-neural autoantibodies (3×anti-CASPR2, 3× anti-NM DAR, 3× anti-LGI1, 3× anti-Hu, 3× anti-Ri, 2×anti-Yo/anti-Ri, 3× anti-Yo, 3× anti-AQP4, 10× anti-GAD65), and 10 serawith a similar staining pattern as P1 and P2 without known autoantibodyreactivity.

Indirect Immunofluorescence Assay (IFA)

IFA was conducted using slides with a biochip array of brain tissuecryosections (hippocampus of rat, cerebellum of rat and monkey) combinedwith recombinant HEK293 cells separately expressing 30 different brainantigens Hu, Yo, Ri, CV2, PNMA2, ITPR1, Homer 3, CARP VIII, ARHGAP26,ZIC4, DNER/Tr, GAD65, GAD67, amphiphysin, recoverin, GABA_(B) receptor,glycine receptor, DPPX, IgLON5, glutamate receptors (types NMDA, AMPA,mGluR1, mGluR5, GLURD2), LGI1, CASPR2, AQP4 (M1 and M23), MOG, ATP1A3,NCDN (EUROIMMUN, FA 111a-1003-51, FA 1112-1003-50, FA-1128-1003-50,FA112d-1003-1, FA 112m-1003-50, FA 1151-1003-50, Miske R, Hahn S,Rosenkranz T, Müller M, Dettmann I M, Mindorf S, Denno Y, Brakopp S,Scharf M, Teegen B, Probst C, Melzer N, Meinck H M, Terborg C, StöckerW, Komorowski L., 2016, Autoantibodies against glutamate receptor δ2after allogenic stem cell transplantation. Neurol NeuroimmunolNeuroinflamm., 3(4):e255; Scharf M, Miske R, Heidenreich F, Giess R,Landwehr P, Blöcker IM, Begemann N, Denno Y, Tiede S, Dähnrich C,Schlumberger W, Unger M, Teegen B, Stöcker W, Probst C, Komorowski L,2015, Neuronal Na+/K+ATPase is an autoantibody target in paraneoplasticneurologic syndrome, Neurology; 84(16):1673-9; Miske R, Gross C C,Scharf M, Golombeck K S, Hartwig M, Bhatia U, Schulte-Mecklenbeck A,Bönte K, Strippel C, Schöls L, Synofzik M, Lohmann H, Dettmann I M,Deppe M, Mindorf S, Warnecke T, Denno Y, Teegen B, Probst C, Brakopp S,Wandinger K P, Wiendl H, Stöcker W, Meuth S G, Komorowski L, Melzer N,2016, Neurochondrin is a neuronal target antigen in autoimmunecerebellar degeneration, Neurol Neuroimmunol Neuroinflamm.; 4(1):e307)).Each biochip mosaic was incubated with 70 μL of PBS-diluted sample atroom temperature for 30 min, washed with PBS-Tween and immersed inPBS-Tween for 5 min. In the second step, either Alexa488-labelled goatanti-human IgG (Jackson Research, Suffolk, United Kingdom), orfluorescein isothiocyanate (FITC)-labelled goat anti-human IgG(EUROIMMUN Medizinische Labordiagnostika AG, Lübeck) were applied andincubated at room temperature for 30 min. Slides were washed again witha flush of PBS-Tween and then immersed in PBS-Tween for 5 min. Slideswere embedded in PBS-buffered, DABCO containing glycerol (approximately20 μL per field) and examined by fluorescence microscopy. Positive andnegative controls were included. Samples were classified as positive ornegative based on fluorescence intensity of the transfected cells indirect comparison with non-transfected cells and control samples.Endpoint titers refer to the last dilution showing visible fluorescence.

Results were evaluated by two independent observers using a EUROSTARIImicroscope (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck,Germany). Reagents were obtained from Merck, Darmstadt, Germany orSigma-Aldrich, Heidelberg, Germany if not specified otherwise.

Immunoblot

Immunoprecipitated cerebellum lysate or lysate of HEK293 cellsexpressing SEQ ID NO: 2 or SEQ-ID 4, or SEQ-ID 5 or SEQ-ID 7 in 0.1%Triton-X-100, 1 mM EDTA buffer, 150 mM NaCl, 100 mM Tris pH 7.4, wereincubated with NuPage LDS sample buffer (ThermoFisher Scientific,Schwerte, Germany) containing 25 mmol/L dithiothreitol at 70° C. for 10minutes, followed by SDS-PAGE (NuPAGE, ThermoFisher Scientific,Schwerte, Germany). Separated proteins were electrotransferred onto anitrocellulose membrane by tank blotting with transfer buffer(ThermoFisher Scientific) according to the manufacturer's instructions.The membranes were blocked with Universal Blot Buffer plus (EUROIMMUNMedizinische Labordiagnostika AG, Lübeck) for 15 min and incubated withthe patient or control sera (dilution 1:200) or monospecific mouseantibody against STX1B (R+D Systems, MAB6848, 1:10,000) in UniversalBlot Buffer plus for 3 hours, followed by 3 washing steps with UniversalBlot Buffer (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck), asecond incubation for 30 min with anti-human-IgG-AP (EUROIMMUNMedizinische Labordiagnostika AG, Lübeck, 1:10) or anti-mouse-IgG-AP(1:2,000) in Universal Blot Buffer plus, 3 washing steps, and stainingwith NBT/BCIP substrate (EUROIMMUN Medizinische Labordiagnostika AG,Lübeck). Reagents were obtained from Merck, Darmstadt, Germany orSigma-Aldrich, Heidelberg, Germany if not specified otherwise.

Identification of the Antigens

Cerebellum from rat was dissected and shock-frozen in liquid nitrogen.The tissues were homogenised in solubilization buffer (100 mmol/Ltris-HCl pH 7.4, 150 mmol/L sodium chloride, 2.5 mmol/L ethylenediaminetetraacetic acid, 0.5% (w/v) sodium deoxycholate, 1% (w/v) Triton X-100)containing protease inhibitors (Complete mini, Roche Diagnostics,Penzberg, Germany) with a Miccra D-8 (Roth, Karlsruhe, Germany) and ahand homogenizer (Sartorius, Göttingen, Germany) at 4° C. The tissuelysates was centrifuged at 21,000×g at 4° C. for 15 min and clearsupernatants were incubated with patient's serum (diluted 1:16.7) at 4°C. overnight. The samples were then incubated with Protein G Dynabeads(ThermoFisher Scientific, Dreieich, Germany) at 4° C. for 3 h to captureimmunocomplexes. Beads were washed 3 times with PBS, and eluted withNuPage LDS sample buffer (ThermoFisher Scientific, Schwerte, Germany)containing 25 mmol/L dithiothreitol at 70° C. for 10 min.Carbamidomethylation with 59 mM iodoacetamide (Bio-Rad, Hamburg,Germany) was performed prior to SDS-PAGE (NuPAGE, ThermoFisherScientific, Schwerte, Germany). Separated proteins were visualized withCoomassie Brilliant Blue (G-250) (Merck), and identified by massspectrometric analysis.

Mass Spectrometry

Visible protein bands were excised from Coomassie Brilliant Blue G-250stained gels. After destaining and tryptic digestion peptides wereextracted and spotted with α-cyano-4-hydroxycinnamic acid onto a MTPAnchorChip™ 384 TF target.

MALDI-TOF/TOF measurements were performed with an Autoflex III smartbeamTOF/TOF200 System using flexControl 3.4 software. MS spectra for peptidemass fingerprinting (PMF) were recorded in positive ion reflector modewith 4,000-10,000 shots and in a mass range from 600 Da to 4,000 Da.Spectra were calibrated externally with the commercially availablePeptide Calibration Standard II, processed with flexAnalysis 3.4 andpeak lists were analyzed with BioTools 3.2.

The Mascot search engine Mascot Server 2.3 (Matrix Science, London, UK)was used for protein identification by searching against the NCBI orSwissProt database limited to Mammalia. Search parameters were asfollows: Mass tolerance was set to 80 ppm, one missed cleavage site wasaccepted, and carbamidomethylation of cysteine residues as well asoxidation of methionine residues were set as fixed and variablemodifications, respectively. To evaluate the protein hits, asignificance threshold of p<0.05 was chosen.

For further confirmation of the PMF hits two to five peptides of eachidentified protein were selected for MS/MS measurements using the WARPfeedback mechanism of BioTools. Parent and fragment masses were recordedwith 400 and 1000 shots, respectively. Spectra were processed andanalyzed as described above with a fragment mass tolerance of 0.7 Da.

Recombinant Expression of NSF, STX1B, DNM1 and VAMP2 in HEK293

The coding DNAs for human NSF (SEQ ID NO: 1) was obtained by RT-PCR onbrain total RNA and primers ATACGTCTCACATGGCGGGCCGGAGCATGCAAG ([senseNSF], SEQ ID NO: 8) and TATCGTCTCCTCGATCAATCAAAATCAAGGGGGCTAG ([asenseNSF] SEQ ID NO: 9). The amplification products were digested with BsmBIand DpnI. The digested cDNAs were ligated with pTriEx-1 (Merck,Darmstadt, Germany). The resulting construct (SEQ ID NO: 15) coded SEQID NO: 2.

The coding DNAs for human STX1B (SEQ ID NO: 3) was obtained by RT-PCR onbrain total RNA and primers ATACGTCTCACATGAAGGATCGGACTCAAGAGCTGC ([senseSTX1B], SEQ ID NO: 10) and eitherATACGTCTCCTCGAGCTACAAGCCCAGCGTCCCCCCAATG ([asense STX1B], SEQ ID NO: 11)or ATACGTCTCCTCGAGTTTCTTCCTCCGGGCCTTGCTCTG ([asense STX1B(ic)-His], SEQID NO: 12). The amplification products were digested with Esp3l andDpnI. The digested cDNAs were ligated with pTriEx-1 (Merck, Darmstadt,Germany). The resulting constructs (SEQ ID NO: 16 and SEQ ID NO: 17)coded SEQ ID NO: 4 and SEQ ID NO: 5.

The coding DNAs for human VAMP2 (SEQ ID NO: 6) was obtained by RT-PCR onbrain total RNA and primers ATACGTCTCTCATGTCTGCTACCGCTGCCACGGCCC ([senseVAMP2], SEQ ID NO: 13) and ATACGTCTCCTCGAGTTAAGTGCTGAAGTAAACTATGATG([asense VAMP2], SEQ ID NO: 14). The amplification products weredigested with Esp3I and DpnI. The digested cDNAs were ligated withpTriEx-1 (Merck, Darmstadt, Germany). The resulting construct (SEQ IDNO: 18) coded SEQ ID NO: 7.

NSF, STX1B, DNM1 and VAMP2, respectively, were expressed in the humancell line HEK293 after ExGen500-mediated transfection (ThermoFisherScientific) according to the manufacturer's instructions. Cells weretransfected in standard T-flasks and the cells were harvested after 5days. The cell sediment was extracted with solubilization buffer. Theextracts were stored in aliquots at −80° C. until further use.

Characterization of the Patients' Autoantibodies

Indirect immunofluorescence assays (IFA) of sera P1 to P2 usingpermeabilized cryosections of cerebellum showed smooth staining of themolecular and granular layers (FIG. 1). Further monospecific analyseswere conducted with recombinant HEK293 cells expressing 30 neuralautoantigens: Hu, Yo, Ri, CV2, PNMA2, SOX1, ITPR1, Homer 3, CARP VIII,ARHGAP26, ZIC4, DNER/Tr, GAD65, GAD67, amphiphysin, recoverin, GABABreceptor, glycine receptor, DPPX, IgLON5, glutamate receptors (typesNMDA, AMPA, mGluR1, mGluR5, GLURD2), LGI1, CASPR2, AQP4 (M1 and M23),MOG, ATP1A3 and NCDN. No specific reactivity was observed.

Identification of STX1B as the Target Neuronal Autoantigens

The immunoprecipitate from homogenized rat cerebellum obtained with P1and P2 presented a protein of approximately 33 kDa in Coomassie-stainedSDS-PAGE which was absent if the homogenates were incubated with controlsera (FIG. 2A). Using MALDI-TOF MS, the protein was identified as STX1B(UNIPROT acc. # P61265). As a proof for correct antigen identification,immunoprecipitates were tested by Western blot using antibodies againstSTX1B. The immunoprecipitates of the patients' sera contained STX1B asdemonstrated by a 33 kDa band (FIG. 2B). Furthermore, the patients'samples were tested by IFA using transfected HEK293 cells whichexpressed STX1B (SEQ ID NO: 4) (FIG. 3A). Patients' sera and CSFsreacted with the STX1B-expressing cells. In contrast, mock-transfectedcell did not demonstrate any specific antibody binding. Both samplesalso reacted with recombinant His-STX1B in immunoblot usingSTX1B(ic)-His (SEQ ID NO: 5) (FIG. 3B).

The reaction of the patients' auto-antibodies on tissue could beabolished by pre-incubation with HEK293 lysate containing STX1B (SEQ IDNO: 4) (FIG. 3C). Antibody binding was unaffected when a comparablefraction from mock-transfected HEK293 cells was used.

Specificity of Anti-STX1B Auto-Antibodies

Sera from 33 patients with various neural auto-antibody-associatedneurological syndromes (3× anti-CASPR2, 3× anti-NMDAR, 3× anti-LGI1, 3×anti-Hu, 3× anti-Ri, 2× anti-Yo/anti-Ri, 3× anti-Yo, 3× anti-AQP4, 10×anti-GAD65), 10 sera with a similar staining pattern as patient 1 and 2on cerebellum without known anti-neural autoantibody reactivity and 45healthy controls were analyzed by IFA with HEK293-STX1B-His in parallelto the samples of the patients. None of the healthy control seraproduced a similar immunofluorescence pattern as the patients' sera onrat brain tissue, and all were all negative when tested on HEK293 cellsexpressing STX1B. Two of the 10 anti-GAD65 positive sera which werepre-diagnosed with stiff person syndrome (P6, P7) and two (P3, P4) ofthe 10 sera with a similar staining pattern as patient 1 and 2 oncerebellum were positive in IFA and Western blot with recombinant STX1B(FIG. 4).

Reactivity Against Other SNARE Complex Proteins

Screening of anti-STX1B positive and additional sera from patients undersuspicion of having and autoimmune encephalitis characterized byproducing similar IFA patterns as the index sera or sera of patientswith neurological symptoms and defined anti-neural autoantibodies by IFAor Western blot using transfected HEK293 cells recombinantly expressingNSF (SEQ ID NO: 2) and VAMP2 (SEQ ID NO: 7) revealed three anti-NSF (P3,P6, P7) positive (FIG. 5) and one anti-VAMP2 (P5) positive sample (FIG.6B). Mock-transfected cells did not demonstrate any specific antibodybinding. P3 and P5 produced a similar immunofluorescence pattern as theindex patients' sera (P1, P2) on rat brain tissue (FIG. 4A and FIG. 6A).P6 and P7 were anti-GAD65 positive and diagnosed with stiff personsyndrome. The three anti-NSF-positive sera (P3, P6, P7) were alsoanti-STX1B positive (FIGS. 4A-4C). None of the 45 healthy controlsshowed a positive reaction against NSF and VAMP2, respectively.

Immunoprecipitation of Dynamin 1 from the Cerebellum by the Patients'Sera

An immunoprecipitation analysis using the patients' sera and the pigcerebellum lysate was implemented to identify additional targetauto-antigens. The total protein concentration of the pig cerebellumlysate as determined by the BCA assay (section 2.2.2) was ≈20-23 mg/mlduring every preparation.

The analysis was performed by total lysate immunoprecipitation. Theimmunoprecipitated proteins were then resolved by gel electrophoresisand stained with blue silver stain to identify bands unique to the serafrom patients compared with controls, which were subsequently identifiedby MS. An image of a blue silver stained gel following total lysateimmunoprecipitation is shown in FIG. 7.

In this experiment, sera from six patients positive for anti-GAD65 and-GAD67 AAbs compared with two healthy controls were included. Followingstaining of the gel, the pull down of the primary target antigens GAD65and GAD67 at positions ≈65 kDa and ≈67 kDa, respectively, was observedin all patients' sera lanes (FIG. 7, line arrows), but not in the serafrom controls. Additionally, another band unique to the patients' seralanes was identified as dynamin 1 (DNM1) at positions ≈97 kDa, (FIG. 7,elbow arrow).

This result was verified by the second immunoprecipitation method,namely cryo-immunoprecipitation. In this method, the pig cerebellumcryosections were used instead of a tissue lysate. Comparable to theabove method, the immunoprecipitated proteins were resolved in a gel andstained with blue silver stain (FIG. 8). In this experiment, fourpatients' sera positive and one negative for anti-GAD65 and -GAD67 AAbsin addition to three sera from healthy controls were included forrepresentation purposes. The results from the two immunoprecipitationmethods were comparable. A strong pull-down of DNM1 (≈97 kDa) wasobserved in all anti-GAD AAb positive patients' sera (FIG. 8, arrow),except one, wherein the pull-down was weaker (FIG. 8, lane 5).Additionally, there was no pull-down of DNM1 with the healthy controls.The patient serum negative for the pull-down of DNM1 in total lysateimmunoprecipitation (FIG. 7, lane 6) was also negative in this test.

Detection of AAbs Against Cerebellar Enriched DNM1 by Immunoblottingwith the Patients' Sera

IMAC enriched SNARE protein fractions were separated by gelelectrophoresis and transferred onto a nitrocellulose membrane. Resultsof the experiment are shown in FIG. 9. The membrane was cut verticallyinto strips and incubated with an antibody mixture containing anti-DNM1(1:1000), anti-NSF (1:1000), and anti-STX1B (1:2000) (Ab, lane 2), aninternal reference patient serum (1:350) from Euroimmun AG that waspositive for AAbs against GAD, NSF, and STX1B as the positive control(PC, lane 3), and a panel of sera (1:350) from the patients (lanes: 4-8)versus neurological (lanes: 9-12) and healthy (lanes: 13-15) controls.The internal reference serum was immunoreactive against NSF and STX1B inthe cell-based assays (data not shown). Reactivities against DNM1 (≈97kDa), NSF (≈82 kDa), and STX1B (≈33 kDa) were observed with the antibodyand the reference serum (lanes: 2 and 3, red arrows). Furthermore,patients' sera portrayed reactivity against DNM1 (lanes: 4-8). Noneurological controls (lanes: 9-12) or healthy controls, except one(lane 14), portrayed any reactivity against DNM1.

Enriched fractions of DNM1 were resolved in gels and immunoblotted withpatient's sera (n=100) versus neurological (n=65) and healthy (n=70)controls. The relative intensity of each band was normalized againstthat of a reference serum and was expressed as a percentage of theobtained relative intensity. The values were compared by implementingKruskal-Wallis test followed by Dunn's multiple comparisons using theGraph Pad prism 5 software. The reference serum (second upper dot) wasassigned a value of 100 and a cutoff of 3SD above the mean of healthycontrols was calculated (dashed line; relative intensity: ˜15%) forscreening purposes alone. In total, 23 patients' sera in the patientcohort, 0 patients' sera in the neurological control (NC), and 1 subjectin the healthy control (HC), exhibited relative intensity values abovethe cutoff for DNM1 (15%). Therefore, the prevalence of AAbs againstDNM1 is significantly higher in the patient cohort compared with thecontrol groups (***p<0.0001). Graphs represent mean±SD of each group.

Amongst the patients' sera positive for AAbs against DNM1, the number ofpatients' sera positive for anti-GAD AAbs was 15. Remarkably, eightpatients' sera that were negative for anti-GAD AAbs were positive forAAbs against DNM1. Altogether, patients' positive for anti-GAD AAbsmight have a higher prevalence for AAbs against DNM1 compared to thosenegative for anti-GAD AAbs. Values from patients' having no anti-GADAAbs were lower but not negative. With respect to individual disorders,the prevalence of AAbs against DNM1 was higher in patients with SPS,PERM, and cerebellitis compared with other associated movementdisorders.

The invention claimed is:
 1. A method for determining the presence orabsence of an autoantibody that binds to an N-ethylmaleimide sensitivefusion protein (NSF) in a sample of a patient, comprising: (a)contacting the sample of the patient that comprises autoantibodies withthe NSF, and (b) determining the presence or absence of the autoantibodyin the sample.
 2. The method of claim 1, wherein the patient has adisease that is associated with one or more symptoms selected from thegroup consisting of progressive stiffness in truncal muscles,progressive stiffness in proximal leg, rigid gait, lumbar hyperlordosis,chronic pain, spasms in proximal limb and axial muscles, sensitivity totouch and sound, hyperekplexia, myoclonus, depression, anxiety, phobia,fever, headache, confusion, dysarthria, dysphagia, nystagmus,oscillopsia, vertigo, nausea, ataxia, dizziness, seizures, epilepsy andtremor.
 3. The method of claim 1, wherein the sample is a bodily fluidcomprising autoantibodies.
 4. The method of claim 1, wherein step (b)comprises performing a technique or assay selected from the groupconsisting of immunodiffusion techniques, immunoelectrophoretictechniques, light scattering immunoassays, agglutination techniques,labeled immunoassays, radiolabeled immunoassay, enzyme immunoassays,chemiluminscence immunoassays, and immunofluorescence.
 5. The method ofclaim 2, wherein the disease is associated with two or more of thesymptoms.
 6. The method of claim 2, wherein the truncal muscles includethoracolumbar paraspinal muscles, abdominal muscles, or abdominal wallmuscles.
 7. The method of claim 3, wherein the bodily fluid is selectedfrom the group consisting of whole blood, serum, cerebrospinal fluid andsaliva.
 8. The method of claim 4, wherein the enzyme immunoassay isenzyme-linked immunosorbent assay (ELISA).
 9. The method of claim 4,wherein the immunofluorescence is indirect immunofluorescence.
 10. Themethod of claim 2, wherein the disease is a neurological disease. 11.The method of claim 10, wherein the disease is an autoimmune disease ofthe nervous system selected from the group consisting of stiff-personsyndrome, paraneoplastic stiff-person syndrome, progressiveencephalomyelitis with rigidity and myoclonus encephalitis, andencephalitis.